Compositions and methods for production of fermentable sugars

ABSTRACT

The present application provides genetically modified fungal organisms that produce enzyme mixtures exhibiting enhanced hydrolysis of cellulosic material to glucose, enzyme mixtures produced by the genetically modified fungal organisms, and processes for producing glucose from cellulose using such enzyme mixtures.

The present application claims priority to U.S. Prov. Patent Appln. Ser.Nos. 61/409,186, 61/409,217, 61/409,472, and 61/409,480, all of whichwere filed on Nov. 2, 2010, and are hereby incorporated by referenceherein.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file CX35-069US2_ST25.TXT, created onJan. 5, 2012, 72,869 bytes, machine format IBM-PC, MS-Windows operatingsystem, is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention provides compositions and methods for theproduction of fermentable sugars. In some embodiments, the presentinvention provides genetically modified fungal organisms. In someadditional embodiments, the present invention provides enzymes that finduse in enhancing hydrolysis of cellulosic material to fermentable sugars(e.g., glucose), and methods for using the enzymes. In some furtherembodiments, the present invention provides enzyme mixtures useful forthe hydrolysis of cellulosic materials.

BACKGROUND

Cellulose is a polymer of the simple sugar glucose linked by beta-1,4glycosidic bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, andlower overall greenhouse gas production. Wood, agricultural residues,herbaceous crops, and municipal solid wastes have been considered asfeedstocks for ethanol production. These materials primarily consist ofcellulose, hemicellulose, and lignin. Once the cellulose is converted toglucose, the glucose is easily fermented by yeast into ethanol.

Although progress has been made in increasing the efficiency ofenzymatic degradation of lignocellulosic feedstocks, there remains agreat need to improve yield of fermentable sugars using enzymaticprocesses.

SUMMARY OF THE INVENTION

The present invention provides genetically modified fungal organisms, aswell as enzymes that enhance hydrolysis of cellulosic material toglucose, and methods for using the enzymes.

The present invention provides fungal cells that have been geneticallymodified to reduce the amount of endogenous glucose and/or cellobioseoxidizing enzyme activity that is produced by the fungal cells. In someembodiments, the fungal cell is an Ascomycete belonging to thesubdivision Pezizomycotina, and/or wherein the fungal cell is from thefamily Chaetomiaceae. In some embodiments, the fungal cell is a speciesof Myceliophthora, Thielavia, Sporotrichum, Neurospora, Sordaria,Podospora, Magnaporthe, Fusarium, Gibberella, Botryotinia, Humicola,Neosartorya, Pyrenophora, Phaeosphaeria, Sclerotinia, Chaetomium,Nectria, Verticillium, Corynascus, Acremonium, Ctenomyces,Chrysosporium, Scytalidium, Talaromyces, Thermoascus, or Aspergillus. Insome additional embodiments, the fungal cell is a species ofMyceliophthora, Thielavia, Sporotrichum, Chrysosporium, Corynascus,Acremonium, Chaetomium, Ctenomyces, Scytalidium, Talaromyces, orThermoascus, while in some other embodiments, the fungal cell isSporotrichum thermophile Sporotrichum cellulophilum, Thielaviaheterothallica, Thielavia terrestris, Corynascus heterothallicus, orMyceliophthora thermophila. In some embodiments, the fungal cell hasbeen genetically modified to reduce the amount of endogenous glucoseoxidase and/or cellobiose dehydrogenase that is produced by the fungalcell. In some additional embodiments, the fungal cell has beengenetically modified to reduce the amount of endogenous glucose oxidaseand/or cellobiose dehydrogenase that is produced by the fungal cell andto increase the production of at least one saccharide hydrolyzingenzyme. In some further embodiments, the fungal cell has beengenetically modified to reduce the amount of endogenous glucose oxidaseand/or cellobiose dehydrogenase that is produced by the fungal cell andto increase the production of at least one saccharide hydrolyzingenzyme, and wherein the fungal cell is a Basidiomycete belonging to theclass Agaricomycetes. In some embodiments, the Basidiomycete is aspecies of Pleurotus, Peniophora, Trametes, Athelia, Sclerotium,Termitomyces, Flammulina, Coniphora, Ganoderma, Pycnoporus,Ceriporiopsis, Phanerochaete, Gloeophyllum, Hericium, Heterobasidion,Gelatoporia, Lepiota, or Irpex. In some embodiments, the fungal cell hasbeen genetically modified to reduce the amount of the endogenous glucoseoxidase and/or cellobiose dehydrogenase that is secreted by the fungalcell. In some additional embodiments, the fungal cell has beengenetically modified to disrupt the secretion signal peptide of theglucose and/or cellobiose oxidizing enzyme. In some further embodiments,the fungal cell has been genetically modified to reduce the amount ofthe endogenous glucose and/or cellobiose oxidizing enzyme that isexpressed by the fungal cell. In still some additional embodiments, thefungal cell has been genetically modified to disrupt a translationinitiation sequence in the transcript encoding the endogenous glucoseand/or cellobiose oxidizing enzyme. In some additional embodiments, thefungal cell has been genetically modified to introduce a frameshiftmutation in the transcript encoding the endogenous glucose and/orcellobiose oxidizing enzyme. In some further embodiments, the fungalcell has been genetically modified to reduce the transcription level ofa gene encoding the endogenous glucose and/or cellobiose oxidizingenzyme. In some embodiments, the fungal cell has been geneticallymodified to disrupt the promoter of a gene encoding the endogenousglucose and/or cellobiose oxidizing enzyme. In still some additionalembodiments, the fungal cell has been genetically modified to at leastpartially delete at least one gene encoding the endogenous glucoseand/or cellobiose oxidizing enzyme. In some further embodiments, thefungal cell has been genetically modified to reduce the catalyticefficiency of the endogenous glucose and/or cellobiose oxidizing enzyme.In some additional embodiments, the fungal cell has been geneticallymodified to mutate one or more residues in an active site of the glucoseand/or cellobiose oxidizing enzyme. In some further embodiments, thefungal cell has been genetically modified to mutate one or more residuesin a heme binding domain of the glucose and/or cellobiose oxidizingenzyme. In some embodiments of the fungal cells provided herein, theglucose and/or cellobiose oxidizing enzyme is selected from cellobiosedehydrogenase (EC 1.1.99.18), glucose oxidase (EC 1.1.3.4), pyranoseoxidase (EC1.1.3.10), glucooligosaccharide oxidase (EC 1.1.99.B3),pyranose dehydrogenase (EC 1.1.99.29), and glucose dehydrogenase (EC1.1.99.10). In some additional embodiments, the glucose and/orcellobiose oxidizing enzyme comprises an amino acid sequence that is atleast about 80%, about 81%, about 82%, about 83%, about 84%, about 85%,about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,or about 99% identical to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, and/or 16.In some further embodiments, the glucose and/or cellobiose oxidizingenzyme is cellobiose dehydrogenase (EC 1.1.99.18). In some embodiments,the fungal cell has been genetically modified to reduce the amount ofglucose and/or cellobiose oxidizing enzyme activity of two or moreendogenous glucose and/or cellobiose oxidizing enzymes that are producedby the fungal cell prior to genetic modification. In some furtherembodiments, the first of the two or more the glucose and/or cellobioseoxidizing enzymes comprises an amino acid sequence that is at leastabout 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, orabout 99% identical to SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, and/or 16,and a second of the two or more the glucose and/or cellobiose oxidizingenzymes comprises an amino acid sequence that is at least about 80%,about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, and/or 16.

The present invention also provides enzyme mixtures comprising two ormore cellulose hydrolyzing enzymes, wherein at least one of the two ormore cellulose hydrolyzing enzymes is expressed by at least one of thefungal cells provided herein.

The present invention also provides enzyme mixtures comprising two ormore cellulose hydrolyzing enzymes, wherein at least one of the two ormore cellulose hydrolyzing enzymes is produced by a fungal cell that hasbeen genetically modified to reduce the amount of endogenous glucoseand/or cellobiose oxidizing enzyme activity that is secreted by thefungal cell, and wherein the fungal cell is an Ascomycete belonging tothe subdivision Pezizomycotina. In some embodiments, the fungal cell isa species of Myceliophthora, Thielavia, Sporotrichum, Corynascus,Acremonium, Chaetomium, Ctenomyces, Scytalidium, Talaromyces, orThermoascus.

The present invention also provides enzyme mixtures comprising two ormore cellulose hydrolyzing enzymes, wherein at least one of the two ormore cellulose hydrolyzing enzymes is produced by a fungal cell that hasbeen genetically modified to reduce the amount of endogenous glucoseand/or cellobiose oxidizing enzyme activity that is secreted by thefungal cell and to increase the production of at least one saccharidehydrolyzing enzyme, wherein the fungal cell is a Basidiomycete belongingto the class Agaricomycetes.

In some embodiments, the enzyme mixtures are cell-free mixtures. In someadditional embodiments, a substrate of the enzyme mixture comprisespretreated lignocellulose. In some further embodiments, the pretreatedlignocellulose comprises lignocellulose treated by a treatment methodselected from acid pretreatment, ammonium pretreatment, steam explosionand/or organic solvent extraction.

The present invention also provides enzyme mixtures comprising two ormore cellulose hydrolyzing enzymes, wherein the fungal cellulase enzymemixture is modified relative to a parental (or reference) enzyme mixtureto be at least partially deficient in glucose and/or cellobioseoxidizing enzyme activity.

The present invention further provides enzyme mixtures comprising two ormore cellulose hydrolyzing enzymes, at least one of the cellulosehydrolyzing enzymes being endogenous to a fungal cell, wherein thefungal cell is a Basidiomycete belonging to the class Agaricomycetes oran Ascomycete belonging to the subdivision Pezizomycotina and whereinthe enzyme mixture is characterized in that, when the enzyme mixture iscontacted with cellobiose and/or glucose, no more than about 10%, about15% or about 20%, of the cellobiose and/or glucose is oxidized after 10hours.

In some embodiments of the enzyme mixtures, the fungal cell has beengenetically modified to reduce the amount of glucose and/or cellobioseoxidase enzyme activity that is secreted by the fungal cell. In somefurther embodiments, the enzyme mixture is a cell-free mixture. In someadditional embodiments, the enzyme mixture comprises at least onebeta-glucosidase. In some further embodiments, the enzyme mixturecomprises at least one cellulase enzyme selected from endoglucanases(EGs), beta-glucosidases (BGLs), Type 1 cellobiohydrolases (CBH1s), Type2 cellobiohydrolases (CBH2s), and/or glycoside hydrolase 61s (GH61s),and/or variants of the cellulase enzyme. In some embodiments, the enzymemixture further comprises at least one cellobiose dehydrogenase. In someembodiments, the cellobiose dehydrogenase is CDH1 and/or CDH2. In someadditional embodiments, the enzyme mixture further comprises at leastone cellulase enzyme and/or at least one additional enzyme. In somefurther embodiments, the enzyme mixture has been subjected to apurification process to selectively remove one or more glucose and/orcellobiose oxidizing enzymes from the enzyme mixture. In someembodiments, the purification process comprises selective precipitationto separate the glucose and/or cellobiose oxidizing enzymes from otherenzymes present in the enzyme mixture. In some additional embodiments,the enzyme mixtures comprise at least one inhibitor of one or moreglucose and/or cellobiose oxidizing enzymes.

The present invention also provides methods for generating cellobioseand/or glucose comprising contacting a cellulose substrate with anenzyme mixture comprising two or more cellulose hydrolyzing enzymes togenerate glucose and/or cellobiose, wherein at least one of thecellulose hydrolyzing enzymes is endogenous to a fungal cell that is anAscomycete belonging to the subdivision Pezizomycotina, and wherein theenzyme mixture is characterized in that, when the enzyme mixture iscontacted with cellobiose and/or glucose, no more than about 10%, about15%, or about 20% of the cellobiose and/or glucose is oxidized after 10hours. In some embodiments, the Ascomycete is a species ofMyceliophthora, Thielavia, Sporotrichum, Neurospora, Sordaria,Podospora, Magnaporthe, Fusarium, Gibberella, Botryotinia, Humicola,Neosartorya, Pyrenophora, Phaeosphaeria, Sclerotinia, Chaetomium,Nectria, Verticillium, or Aspergillus.

The present invention also provides methods for generating cellobioseand/or glucose comprising contacting a cellulose substrate with anenzyme mixture comprising two or more cellulose hydrolyzing enzymes togenerate glucose and/or cellobiose, wherein at least one of thecellulose hydrolyzing enzymes is endogenous to a fungal cell that is aBasidiomycete belonging to the class Agaricomycetes, and wherein theenzyme mixture is characterized in that, when the enzyme mixture iscontacted with cellobiose and/or glucose, no more than about 10%, about15% or about 20% of the cellobiose and/or glucose is oxidized after 10hours. In some embodiments, the Basidiomycete is a species of Pleurotus,Peniophora, Trametes, Athelia, Sclerotium, Termitomyces, Flammulina,Coniphora, Ganoderma, Pycnoporus, Ceriporiopsis, Phanerochaete,Gloeophyllum, Hericium, Heterobasidion, Gelatoporia, Lepiota, or Irpex.

The present invention also provides methods for generating cellobioseand/or glucose comprising contacting a cellulose substrate with anenzyme mixture comprising two or more cellulose hydrolyzing enzymes togenerate glucose and/or cellobiose, wherein at least one of thecellulose hydrolyzing enzymes is endogenous to a fungal cell that is anAscomycete belonging to the subdivision Pezizomycotina, and wherein, ofthe cellulose hydrolyzed by the enzyme mixture, at least about 80%,about 85%, or about 90% is present in the form of cellobiose and/orglucose. In some embodiments, the Ascomycete is a species ofMyceliophthora, Thielavia, Sporotrichum, Neurospora, Sordaria,Podospora, Magnaporthe, Fusarium, Gibberella, Botryotinia, Humicola,Neosartorya, Pyrenophora, Phaeosphaeria, Sclerotinia, Chaetomium,Nectria, Verticillium, or Aspergillus. In some embodiments, theAscomycete is Myceliophthora thermophila, Thielavia heterothallica orSporotrichum thermophile. In some embodiments, the fungal cell isMyceliophthora thermophila.

The present invention also provides methods for generating cellobioseand/or glucose comprising contacting a cellulose substrate with anenzyme mixture comprising two or more cellulose hydrolyzing enzymes togenerate glucose and/or cellobiose, wherein at least one of thecellulose hydrolyzing enzymes is endogenous to a fungal cell that is aBasidiomycete belonging to the class Agaricomycetes, and wherein, of thecellulose hydrolyzed by the enzyme mixture, at least about 80%, about85%, or about 90% is present in the form of cellobiose and/or glucose.In some embodiments, the Basidiomycete is a species of Pleurotus,Peniophora, Trametes, Athelia, Sclerotium, Termitomyces, Flammulina,Coniphora, Ganoderma, Pycnoporus, Ceriporiopsis, Phanerochaete,Gloeophyllum, Hericium, Heterobasidion, Gelatoporia, Lepiota, or Irpex.

The present invention also provides methods for producing cellobioseand/or glucose from cellulose comprising treating a cellulose substratewith an enzyme mixture to generate glucose, wherein the enzyme mixtureis modified relative to a secreted enzyme mixture from a reference (orparental) fungal cell to be at least partially deficient in glucoseand/or cellobiose oxidizing enzyme activity. In some embodiments of themethods, the enzyme mixture is a cell-free mixture. In some additionalembodiments, the cellulose substrate comprises pretreatedlignocellulose. In some further embodiments, the pretreatedlignocellulose comprises lignocellulose treated by a treatment methodselected from acid pretreatment, ammonium pretreatment, steam explosionand/or organic solvent extraction. In some further embodiments, themethods further comprise fermentation of the cellobiose and/or glucoseto at least one end product. In some embodiments, the end product is atleast one fuel alcohol and/or at least one precursor industrialchemical. In some additional embodiments, the fuel alcohol is ethanol orbutanol. In some embodiments, the process for producing cellobioseand/or glucose from cellulose and said fermentation are conducted in asimultaneous saccharification and fermentation (SSF) process. In somefurther additional embodiments, the enzyme mixture is produced by afungal cell has that been genetically modified to reduce the amount ofone or more endogenous glucose and/or cellobiose oxidizing enzymes thatis secreted by the fungal cell. In some embodiments, the enzyme mixturehas been subjected to a purification process to selectively remove atleast one glucose and/or cellobiose oxidizing enzyme from the enzymemixture. In some further embodiments, the purification process comprisesselective precipitation to separate the glucose and/or cellobioseoxidizing enzyme from other enzymes present in the enzyme mixture. Instill some additional embodiments, the enzyme mixture comprises at leastone inhibitor of the glucose and/or cellobiose oxidizing enzyme. In someembodiments, the inhibitor comprises a broad-spectrum oxidase inhibitorselected from sodium azide, potassium cyanide, a metal anion, and acombination thereof. In some embodiments, the inhibitor comprises aspecific inhibitor of cellobiose dehydrogenase (EC 1.1.99.18) selectedfrom cellobioimidazole, gentiobiose, lactobiono-1,5-lactone,celliobono-1,5-lactone, tri-N-acetylchitortriose, methyl-beta-Dcellobiosidase, 2,2-bipyridine, cytochrome C, and a combination thereof.In some embodiments, the method is a batch process, while in some otherembodiments it is a continuous process, and in some further embodimentsit is a fed-batch process and in still further embodiments, it is acombination of batch, continuous and/or fed-batch processes conducted inany order. In some embodiments, the method is conducted in a reactionvolume of at least 10,000 liters, while in some other embodiments, themethod is conducted in a reaction volume of at least 100,000 liters. Insome embodiments, the enzyme mixture comprises at least onebeta-glucosidase, while in some other embodiments, the enzyme mixturedoes not comprise a beta-glucosidase. In some embodiments, the enzymemixture comprises at least one endoglucanase, while in some otherembodiments, the enzyme mixture does not comprise an endoglucanase. Insome embodiments, the enzyme mixture comprises at least one cellulaseenzyme selected from endoglucanases (EGs), beta-glucosidases (BGLs),Type 1 cellobiohydrolases (CBH1s), Type 2 cellobiohydrolases (CBH2s),and/or glycoside hydrolase 61s (GH61s), and/or variants of saidcellulase enzyme.

The present invention also provides methods for generating glucosecomprising contacting cellulose with an enzyme mixture comprising two ormore cellulose hydrolyzing enzymes, wherein at least one of the two ormore cellulose hydrolyzing enzymes is produced by the fungal cellsprovided herein.

The present invention also provides methods for generating glucosecomprising contacting cellulose with an enzyme mixture comprising two ormore cellulose hydrolyzing enzymes, wherein at least one of the two ormore cellulose hydrolyzing enzymes is produced by a fungal cell that hasbeen genetically modified to reduce the amount of endogenous glucoseand/or cellobiose oxidizing enzyme activity that is secreted by thefungal cell, wherein the fungal cell is an Ascomycete belonging to thesubdivision Pezizomycotina. In some embodiments, the fungal cell is aspecies of Myceliophthora, Thielavia, Sporotrichum, Corynascus,Acremonium, Chaetomium, Ctenomyces, Scytalidium, Talaromyces, orThermoascus.

The present invention also provides methods for generating glucosecomprising contacting cellulose with an enzyme mixture comprising two ormore cellulose hydrolyzing enzymes, wherein at least one of the two ormore cellulose hydrolyzing enzymes is produced by a fungal cell that hasbeen genetically modified to reduce the amount of endogenous glucoseand/or cellobiose oxidizing enzyme activity that is secreted by thefungal cell and to increase the production of at least one saccharidehydrolyzing enzyme, wherein the fungal cell is a Basidiomycete belongingto the class Agaricomycetes.

The present invention further provides methods for generating glucosecomprising contacting cellulose with at least one enzyme mixture asprovided herein. In some embodiments, the cellulose comprises pretreatedlignocellulose. In some additional embodiments, the pretreatedlignocellulose comprises lignocellulose treated by a treatment methodselected from acid pretreatment, ammonium pretreatment, steam explosionand/or organic solvent extraction. In some additional embodiments, theenzyme mixture is a cell-free mixture. In some further embodiments, themethods further comprise fermentation of the glucose to an end product.In some embodiments, the end product is a fuel alcohol or a precursorindustrial chemical. In some embodiments, the fuel alcohol is ethanol orbutanol.

The present invention further provides the fungal cells provided herein,as well as the enzyme mixtures provided herein, and the methods providedherein, further comprising a cellulose degrading enzyme that isheterologous to the fungal cell.

The present invention also provides fermentation media comprising atleast one fungal cell provided herein.

The present invention also provides fermentation media comprising atleast one enzyme mixture provided herein.

The present invention further provides fermentation media comprising atleast one fungal cell and/or at least one enzyme mixture, as providedherein.

The present invention also provides methods of producing at least onecellulase, comprising at least one fungal cell provided herein, underconditions such that said at least one cellulase is produced. In someembodiments, the fungal cell is recombinant.

The present invention also provides compositions comprising at least onecellulase as provided herein.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart that shows the products of cellulose hydrolysis usingenzyme mixtures obtained from strains CF-402, CF-403, and CF-401 asfurther described in Example 1 and Example 7. Dark bars representmeasured glucose production. Light bars represent measured gluconateproduction. Numbers above horizontal bars indicate the sum of glucoseand gluconate fractions.

FIG. 2 is a chart that shows the products of cellulose hydrolysis usingenzyme mixtures produced by strain CF-400 (comprising a cdh1 deletion);strain CF-401 (comprising the deletions of cdh1 and cdh2) and strainCF-402 (comprising cdh1 and cdh2), as further described in Example 8.

FIGS. 3 and 4 provide the nucleotide and amino acid sequences of M.thermophila CDH1 and CDH2 (SEQ ID NOS:5-8).

FIGS. 5 and 6 provide the nucleotide and amino acid sequences of M.thermophila GO1 and GO2 (SEQ ID NOS:1-4).

FIG. 7 provides the nucleotide and amino acid sequences of A. oryzaepyranose oxidase (SEQ ID NOS:9-10).

FIG. 8 provides the nucleotide and amino acid sequences of A. strictumglucooligosaccharide oxidase (SEQ ID NOS:11-12).

FIG. 9 provides the nucleotide and amino acid sequences of A. bisporuspyranose dehydrogenase (SEQ ID NOS:13-14).

FIG. 10 provides the nucleotide and amino acid sequences of T.stipitatus ATCC10500 glucose dehydrogenase (SEQ ID NOS:15-16).

FIG. 11 provides a chart showing fractional recovery of availablecellulose using an enzyme mixture containing cellobiose dehydrogenaseactivity. Dark bars represent glucose yield as measured using ahorseradish peroxidase coupled enzymatic assay described in Example 1.Light bars represent expected glucose yield calculated using the IRmethod for determining cellulose conversion described in Example 9.

FIGS. 12A and 12B are HPLC chromatograms showing the effect of acidhydrolysis of cellotriose (FIG. 12A) or of cellulose hydrolysis productsproduced by an enzyme mixture containing cellobiose dehydrogenase (FIG.12B) as described in Example 11.

FIG. 13 provides an IR spectrum of cellulose hydrolysate obtained usingenzyme mixtures lacking (Turbo) or containing (CF-402) cellobiosedehydrogenase activity. The vertical arrow indicates the carbonyl peakat 1715 cm⁻¹ unique to the hydrolysate produced by the CF-402 enzymemixture.

FIGS. 14A and 14B are HPLC chromatograms that identify an oxidizedglucose product produced from glucose (FIG. 5A) or from cellulosehydrolysate using cellulase enzymes secreted by strain CF-402, asdescribed in Example 13.

DESCRIPTION OF THE INVENTION

The present invention provides genetically modified fungal organisms, aswell as enzymes that enhance hydrolysis of cellulosic material toglucose, and methods for using the enzymes.

All patents and publications, including all sequences disclosed withinsuch patents and publications, referred to herein are expresslyincorporated by reference. Unless otherwise indicated, the practice ofthe present invention involves conventional techniques commonly used inmolecular biology, fermentation, microbiology, and related fields, whichare known to those of skill in the art. Unless defined otherwise herein,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. Although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention, some suitable methods and materialsare described. Indeed, it is intended that the present invention not belimited to the particular methodology, protocols, and reagents describedherein, as these may vary, depending upon the context in which they areused. The headings provided herein are not limitations of the variousaspects or embodiments of the present invention.

Nonetheless, in order to facilitate understanding of the presentinvention, a number of terms are defined below. Numeric ranges areinclusive of the numbers defining the range. Thus, every numerical rangedisclosed herein is intended to encompass every narrower numerical rangethat falls within such broader numerical range, as if such narrowernumerical ranges were all expressly written herein. It is also intendedthat every maximum (or minimum) numerical limitation disclosed hereinincludes every lower (or higher) numerical limitation, as if such lower(or higher) numerical limitations were expressly written herein.

As used herein, the term “comprising” and its cognates are used in theirinclusive sense (i.e., equivalent to the term “including” and itscorresponding cognates).

As used herein and in the appended claims, the singular “a”, “an” and“the” include the plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to a “host cell” includes aplurality of such host cells.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. The headings provided hereinare not limitations of the various aspects or embodiments of theinvention that can be had by reference to the specification as a whole.Accordingly, the terms defined below are more fully defined by referenceto the specification as a whole.

As used herein, “substrate” refers to a substance or compound that isconverted or meant to be converted into another compound by the actionof an enzyme. The term includes not only a single compound but alsocombinations of compounds, such as solutions, mixtures and othermaterials which contain at least one substrate.

As used herein, “conversion” refers to the enzymatic transformation of asubstrate to the corresponding product. “Percent conversion” refers tothe percent of the substrate that is converted to the product within aperiod of time under specified conditions. Thus, for example, the“enzymatic activity” or “activity” of a cellobiose dehydrogenase (“CDH”or “cdh”) polypeptide can be expressed as “percent conversion” of thesubstrate to the product.

As used herein, “secreted activity” refers to enzymatic activity ofglucose and/or cellobiose oxidizing enzymes produced by a fungal cellthat is present in an extracellular environment. An extracellularenvironment can be, for example, an extracellular milieu such as aculture medium. The secreted activity is influenced by the total amountof glucose and/or cellobiose oxidizing enzyme secreted, and also isinfluenced by the catalytic efficiency of the secreted glucose and/orcellobiose oxidizing enzyme.

As used herein, a “reduction in catalytic efficiency” refers to areduction in the activity of the glucose and/or cellobiose oxidizingenzyme, relative to unmodified glucose and/or cellobiose oxidizingenzyme, as measured using standard techniques, as provided herein orotherwise known in the art.

As used herein, the term “enzyme mixture” refers to a combination of atleast two enzymes. In some embodiments, at least two enzymes are presentin a composition. In some additional embodiments, the enzyme mixturesare present within a cell (e.g., a fungal cell). In some embodiments,each or some of the enzymes present in an enzyme mixture are produced bydifferent fungal cells and/or different fungal cultures. In some furtherembodiments, all of the enzymes present in an enzyme mixture areproduced by the same cell. In some embodiments, the enzyme mixturescomprise cellulase enzymes, while in some additional embodiments, theenzyme mixtures comprise enzymes other than cellulases. In someembodiments, the enzyme mixtures comprise at least one cellulase and atleast one enzyme other than a cellulase. In some embodiments, the enzymemixtures comprise enzymes including, but not limited to endoxylanases(EC 3.2.1.8), beta-xylosidases (EC 3.2.1.37),alpha-L-arabinofuranosidases (EC 3.2.1.55), alpha-glucuronidases (EC3.2.1.139), acetylxylanesterases (EC 3.1.1.72), feruloyl esterases (EC3.1.1.73), coumaroyl esterases (EC 3.1.1.73), alpha-galactosidases (EC3.2.1.22), beta-galactosidases (EC 3.2.1.23), beta-mannanases (EC3.2.1.78), beta-mannosidases (EC 3.2.1.25), endo-polygalacturonases (EC3.2.1.15), pectin methyl esterases (EC 3.1.1.11), endo-galactanases (EC3.2.1.89), pectin acetyl esterases (EC 3.1.1.6), endo-pectin lyases (EC4.2.2.10), pectate lyases (EC 4.2.2.2), alpha rhamnosidases (EC3.2.1.40), exo-galacturonases (EC 3.2.1.82), exo-galacturonases (EC3.2.1.67), exopolygalacturonate lyases (EC 4.2.2.9), rhamnogalacturonanendolyases EC (4.2.2.B3), rhamnogalacturonan acetylesterases (EC3.2.1.B11), rhamnogalacturonan galacturonohydrolases (EC 3.2.1.B11),endo-arabinanases (EC 3.2.1.99), laccases (EC 1.10.3.2),manganese-dependent peroxidases (EC 1.10.3.2), amylases (EC 3.2.1.1),glucoamylases (EC 3.2.1.3), lipases, lignin peroxidases (EC 1.11.1.14),and/or proteases.

In some additional embodiments, the present invention further providesenzyme mixtures comprising at least one expansin and/or expansin-likeprotein, such as a swollenin (See e.g., Salheimo et al., Eur. J.Biochem., 269:4202-4211 [2002]) and/or a swollenin-like protein.Expansins are implicated in loosening of the cell wall structure duringplant cell growth. Expansins have been proposed to disrupt hydrogenbonding between cellulose and other cell wall polysaccharides withouthaving hydrolytic activity. In this way, they are thought to allow thesliding of cellulose fibers and enlargement of the cell wall. Swollenin,an expansin-like protein contains an N-terminal Carbohydrate BindingModule Family 1 domain (CBD) and a C-terminal expansin-like domain. Insome embodiments, an expansin-like protein and/or swollenin-like proteincomprises one or both of such domains and/or disrupts the structure ofcell walls (e.g., disrupting cellulose structure), optionally withoutproducing detectable amounts of reducing sugars. In some additionalembodiments, the enzyme mixtures comprise at least one polypeptideproduct of a cellulose integrating protein, scaffoldin and/or ascaffoldin-like protein (e.g., CipA or CipC from Clostridiumthermocellum or Clostridium cellulolyticum respectively). In someadditional embodiments, the enzyme mixtures comprise at least onecellulose induced protein and/or modulating protein (e.g., as encoded bycip1 or cip2 gene and/or similar genes from Trichoderma reesei; Seee.g., Foreman et al., J. Biol. Chem., 278:31988-31997 [2003]). In someadditional embodiments, the enzyme mixtures comprise at least one memberof each of the classes of the polypeptides described above, severalmembers of one polypeptide class, or any combination of thesepolypeptide classes to provide enzyme mixtures suitable for varioususes.

Any combination of at least one two, three, four, five, or more thanfive enzymes and/or polypeptides find use in various enzyme mixturesprovided herein. Indeed, it is not intended that the enzyme mixtures ofthe present invention be limited to any particular enzymes,polypeptides, nor combinations, as any suitable enzyme mixture finds usein the present invention.

As used herein, the term “saccharide” refers to any carbohydratecomprising monosaccharides (e.g., glucose, ribose, fructose, galactose,etc.), disaccharides (e.g., sucrose, lactose, maltose, cellobiose,trehalose, melibiose, etc.), oligosaccharides (e.g., raffinose,stachyose, amylose, etc.), and polysaccharides (e.g., starch, glycogen,cellulose, chitin, xylan, arabinoxylan, mannan, fucoidan, galactomannan,callose, laminarin, chrysolaminarin, amylopectin, dextran, dextrins,maltodextrins, inulin, oligofructose, polydextrose, etc.). The termencompasses simple carbohydrates, as well as complex carbohydrates.Indeed, it is not intended that the present invention be limited to anyparticular saccharide, as various saccharides and forms of saccharidesfind use in the present invention.

As used herein, the term “saccharide hydrolyzing enzyme” refers to anyenzyme that hydrolyzes at least one saccharide.

As used herein, the terms “glucose oxidizing enzyme” and “cellobioseoxidizing enzyme” refer to enzymes that oxidize glucose and/orcellobiose. For example, glucose and/or cellobiose oxidizing enzymesinclude glucose oxidase (EC 1.1.3.4), cellobiose dehydrogenase (EC1.1.99.18), pyranose oxidase (EC 1.1.3.10), glucooligosaccharide oxidase(EC 1.1.99.B3), pyranose dehydrogenase (EC 1.1.99.29), and glucosedehydrogenase (EC 1.1.99.10).

As used herein, the terms “glucose oxidase” and “GO” refer to an enzymethat is an oxido-reductase that catalyses the oxidation of β-D-glucoseinto D-glucono-1,5-lactone, which is a cyclic ester existing at apH-dependent equilibrium in aqueous solution with gluconic acid orgluconate. Exemplary glucose oxidases fall into the enzymeclassification (EC 1.1.3.4). In order to work as a catalyst, glucoseoxidases typically utilize a co-substrate oxidant, such as flavinadenine dinucleotide (FAD). The enzyme is highly specific forβ-D-glucose. However, glucose oxidase also can demonstrate some lesseroxidase activity for substrates 2-deoxy-D-glucose, D-mannose andD-galactose (See e.g., Bentley, Meth. Enzymol., 9:86 [1996]).

As used herein, the terms “cellobiose dehydrogenase” and “CDH” refer toa cellobiose:acceptor 1-oxidoreductase that catalyzes the conversion ofcellobiose in the presence of an acceptor to cellobiono-1,5-lactone anda reduced acceptor. Examples of cellobiose dehydrogenases fall into theenzyme classification (E.C. 1.1.99.18). Typically 2,6-Dichloroindophenolcan act as acceptor, as can iron, especially Fe(SCN)₃, molecular oxygen,ubiquinone, or cytochrome C, and other polyphenolics, such as lignin.Substrates of the enzyme include cellobiose, cello-oligosaccharides,lactose, and D-glucosyl-1,4-β-D-mannose, glucose, maltose, mannobiose,thiocellobiose, galactosyl-mannose, xylobiose, xylose. Electron donorsinclude beta-1-4 dihexoses with glucose or mannose at the reducing end,though alpha-1-4 hexosides, hexoses, pentoses, and beta-1-4 pentomerscan act as substrates for at least some of these enzymes (See e.g.,Henriksson et al, Biochim. Biophys. Acta—Prot. Struct. Mol. Enzymol.,1383: 48-54 [1998]; and Schou et al., Biochem. J., 330: 565-571 [1998]).

As used herein, the terms “oxidation”, “oxidize(d)” and the like as usedherein refer to the enzymatic formation of one or more glucose orcellobiose oxidation products including, but not limited to,cellobionolactone, cellobionic acid, gluconolactone, gluconate and/orgluconic acid. When used in reference to a percentage of oxidizedcellobiose and/or glucose, those percentages reflect a weight percent(w/w) relative to the initial amount of substrate. For example, when theenzyme mixture is contacted with cellobiose and/or glucose, thepercentage of oxidized cellobiose and/or glucose reflects a weightpercent (w/w) relative to the initial amount of cellobiose and/orglucose present in solution. Where the enzyme mixture is contacted witha cellulose substrate, the percentage of oxidized cellobiose and/orglucose reflects a weight percent (w/w) based on the maximum amount (wt%) of glucose that could be produced from the total hydrolyzed cellulose(i.e., Gmax).

As used herein, the terms “cellobiose dehydrogenase” and “CDH” refer toa cellobiose:acceptor 1-oxidoreductase that catalyzes the conversion ofcellobiose in the presence of an acceptor to cellobiono-1,5-lactone anda reduced acceptor. Examples of cellobiose dehydrogenases are includedin the enzyme classification (E.C. 1.1.99.18). In some embodiments, thecellobiose dehydrogenase of interest in the present invention is CDH1,which is encoded by the cdh1 gene. In some embodiments, the cellobiosedehydrogenase of interest in the present invention is CDH2, which isencoded by the cdh2 gene. In some embodiments, both CDH1 and CDH2 are ofinterest.

As used herein, the terms “pyranose oxidase” and “PO” refer to an enzymethat catalyzes the conversion of D-glucose and O₂ to 2-dehydro-D-glucoseand H₂O₂. Examples of pyranose oxidases fall into the enzymeclassification (E.C. 1.1.3.10). The systematic name of this enzyme classis pyranose:oxygen 2-oxidoreductase. Other names in common use includeglucose 2-oxidase, and pyranose-2-oxidase. Substrates of the enzymeinclude D-glucose, D-xylose, L-arabinose, L-sorbose,D-glucono-1,5-lactone, cellobiose and gentiobiose.

As used herein, the terms “glucooligosaccharide oxidase” and “GOOX”refer to an enzyme that catalyzes the oxidation of oligosaccharides withglucose on the reducing end and each sugar residue joined by an alpha-or beta-1,4 glucosidic bond. Examples of glucooligosaccharide oxidasefall into the enzyme classification (E.C. 1.1.99.B3). The systematicname of this enzyme class is carbohydrate:acceptor oxidoreductase.Substrates of the enzyme include maltose, lactose, cellobiose andmaltose derivatives up to seven residues.

As used herein, the terms “pyranose dehydrogenase” and “PDH” refer to anenzyme that catalyzes the reaction of pyranose and an acceptor to yield2-dehydropyranose (or 3-dehydropyranose or 2,3-didehydropyranose) and areduced acceptor. PDH also catalyzes the reaction of a pyranoside and anacceptor to yield a 3-dehydropyranoside (or 3,4-didehydropyranoside) anda reduced acceptor. Examples of pyranose dehydrogenases fall into theenzyme classification (E.C. 1.1.99.29). The systematic name of thisenzyme class is pyranose:acceptor oxidoreductase. Other names in commonuse include pyranose 2,3-dehydrogenase. PDH utilizes FAD as a cofactor.A number of aldoses and ketoses in pyranose form, as well as glycosides,gluco-oligosaccharides, sucrose and lactose can act as a donor.1,4-Benzoquinone or ferricenium ion (ferrocene oxidized by removal ofone electron) can serve as acceptor. Unlike EC 1.1.3.10 (pyranoseoxidase), pyranose dehydrogenase does not interact with O₂ and exhibitsextremely broad substrate tolerance with variable regioselectivity (C-3,C-2 or C-3+C-2 or C-3+C-4) for (di)oxidation of different sugars.D-Glucose is exclusively or preferentially oxidized at C-3 (depending onthe enzyme source), but can also be oxidized at C-2+C-3. Pyranosedehydrogenase also acts on 1->4-alpha- and1->4-beta-gluco-oligosaccharides, non-reducing gluco-oligosaccharidesand L-arabinose, which are not substrates of EC 1.1.3.10. Sugars areoxidized by pyranose dehydrogenase in their pyranose but not in theirfuranose form.

As used herein, the terms “glucose dehydrogenase” and “GDH” refer to anenzyme that catalyzes the reaction of D-glucose and an acceptor to yieldD-glucono-1,5-lactone and a reduced acceptor. Examples of glucosedehydrogenase fall into the enzyme classification (E.C. 1.1.99.10). Thesystematic name of this enzyme class is D-glucose:acceptor1-oxidoreductase. GDH utilizes FAD as a cofactor.

As used herein, the term “cellulase” refers to any enzyme that iscapable of degrading cellulose. Thus, the term encompasses enzymescapable of hydrolyzing cellulose (β-1,4-glucan or β-D-glucosidiclinkages) to shorter cellulose chains, oligosaccharides, cellobioseand/or glucose. “Cellulases” are divided into three sub-categories ofenzymes: 1,4-β-D-glucan glucanohydrolase (“endoglucanase” or “EG”);1,4-β-D-glucan cellobiohydrolase (“exoglucanase,” “cellobiohydrolase,”or “CBH”); and β-D-glucoside-glucohydrolase (“β-glucosidase,”“cellobiase,” “BG,” or “BGL”). These enzymes act in concert to catalyzethe hydrolysis of cellulose-containing substrates. Endoglucanases breakinternal bonds and disrupt the crystalline structure of cellulose,exposing individual cellulose polysaccharide chains (“glucans”).Cellobiohydrolases incrementally shorten the glucan molecules, releasingmainly cellobiose units (a water-soluble β-1,4-linked dimer of glucose)as well as glucose, cellotriose, and cellotetrose. Beta-glucosidasessplit the cellobiose into glucose monomers.

Cellulases often comprise a mixture of different types of cellulolyticenzymes (endoglucanases and cellobiohydrolases) that act synergisticallyto break down the cellulose to soluble di- or oligosaccharides such ascellobiose, which are then further hydrolyzed to glucose bybeta-glucosidase. Cellulase enzymes are produced by a wide variety ofmicroorganisms. Cellulases (and hemicellulases) from filamentous fungiand some bacteria are widely exploited for many industrial applicationsthat involve processing of natural fibers to sugars.

As used herein, a “cellulase-producing fungal cell” is a fungal cellthat produces at least one cellulase enzyme (i.e., “cellulosehydrolyzing enzyme”). In some embodiments, the cellulase-producingfungal cells provided herein express and secrete a mixture of cellulosehydrolyzing enzymes. As used herein, the terms “cellulose hydrolyzingenzyme,” “cellulolytic enzyme,” and like terms refer to an enzyme thatacts in the process of breaking down cellulose to soluble di- oroligosaccharides such as cellobiose, which are then further hydrolyzedto glucose by beta-glucosidase. A mixture of cellulose hydrolyzingenzymes is also referred to herein as “cellulases,” a“cellulase-containing mixture,” and/or a “cellulase mixture.”

As used herein, the terms “endoglucanase” and “EG” refer to a categoryof cellulases (EC 3.2.1.4) that catalyze the hydrolysis of internalβ-1,4 glucosidic bonds of cellulose. The term “endoglucanase” is furtherdefined herein as an endo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase(E.C. 3.2.1.4), which catalyses endohydrolysis of 1,4-beta-D-glycosidiclinkages in cellulose, cellulose derivatives (such as carboxymethylcellulose and hydroxyethyl cellulose), lichenan, beta-1,4 bonds in mixedbeta-1,3 glucans such as cereal beta-D-glucans or xyloglucans, and otherplant material containing cellulosic components. Endoglucanase activitycan be determined based on a reduction in substrate viscosity orincrease in reducing ends determined by a reducing sugar assay (Seee.g., Zhang et al., Biotechnol. Adv., 24:452-481 [2006]). For purposesof the present invention, endoglucanase activity is determined usingcarboxymethyl cellulose (CMC) hydrolysis (See e.g., Ghose, Pur. Appl.Chem., 59:257-268 [1987]).

As used herein, “EG1” refers to a carbohydrate active enzyme expressedfrom a nucleic acid sequence coding for a glycohydrolase (GH) Family 7catalytic domain classified under EC 3.2.1.4 or any protein, polypeptideor catalytically active fragment thereof. In some embodiments, the EG1is functionally linked to a carbohydrate binding module (CBM), such as aFamily 1 cellulose binding domain.

As used herein, the term “EG2” refers to a carbohydrate active enzymeexpressed from a nucleic acid sequence coding for a glycohydrolase (GH)Family 5 catalytic domain classified under EC 3.2.1.4 or any protein,polypeptide or catalytically active fragment thereof. In someembodiments, the EG2 is functionally linked to a carbohydrate bindingmodule (CBM), such as a Family 1 cellulose binding domain.

As used herein, the term “EG3” refers to a carbohydrate active enzymeexpressed from a nucleic acid sequence coding for a glycohydrolase (GH)Family 12 catalytic domain classified under EC 3.2.1.4 or any protein,polypeptide or catalytically active fragment thereof. In someembodiments, the EG3 is functionally linked to a carbohydrate bindingmodule (CBM), such as a Family 1 cellulose binding domain.

As used herein, the term “EG4” refers to a carbohydrate active enzymeexpressed from a nucleic acid sequence coding for a glycohydrolase (GH)Family 61 catalytic domain classified under EC 3.2.1.4 or any protein,polypeptide or fragment thereof. In some embodiments, the EG4 isfunctionally linked to a carbohydrate binding module (CBM), such as aFamily 1 cellulose binding domain.

As used herein, the term “EG5” refers to a carbohydrate active enzymeexpressed from a nucleic acid sequence coding for a glycohydrolase (GH)Family 45 catalytic domain classified under EC 3.2.1.4 or any protein,polypeptide or fragment thereof. In some embodiments, the EG5 isfunctionally linked to a carbohydrate binding module (CBM), such as aFamily 1 cellulose binding domain.

As used herein, the term “EG6” refers to a carbohydrate active enzymeexpressed from a nucleic acid sequence coding for a glycohydrolase (GH)Family 6 catalytic domain classified under EC 3.2.1.4 or any protein,polypeptide or fragment thereof. In some embodiments, the EG6 isfunctionally linked to a carbohydrate binding module (CBM), such as aFamily 1 cellulose binding domain.

As used herein, the terms “cellobiohydrolase” and “CBH” refer to acategory of cellulases (EC 3.2.1.91) that hydrolyze glycosidic bonds incellulose. The term “cellobiohydrolase” is further defined herein as a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91), which catalyzes thehydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellooligosaccharides, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing ends ofthe chain (See e.g., Teeri, Tr. Biotechnol., 15:160-167 [1997]; andTeeri et al., Biochem. Soc. Trans., 26:173-178 [1998]). In someembodiments, cellobiohydrolase activity is determined using afluorescent disaccharide derivative4-methylumbelliferyl-.beta.-D-lactoside (See e.g., van Tilbeurgh et al.,FEBS Lett., 149:152-156 [1982]; and van Tilbeurgh and Claeyssens, FEBSLett., 187:283-288 [1985]).

As used herein, the terms “CBH1” and “type 1 cellobiohydrolase” refer toa carbohydrate active enzyme expressed from a nucleic acid sequencecoding for a glycohydrolase (GH) Family 7 catalytic domain classifiedunder EC 3.2.1.91 or any protein, polypeptide or catalytically activefragment thereof. In some embodiments, the CBH1 is functionally linkedto a carbohydrate binding module (CBM), such as a Family 1 cellulosebinding domain.

As used herein, the terms “CBH2” and “type 2 cellobiohydrolase” refer toa carbohydrate active enzyme expressed from a nucleic sequence codingfor a glycohydrolase (GH) Family 6 catalytic domain classified under EC3.2.1.91 or any protein, polypeptide or catalytically active fragmentthereof. Type 2 cellobiohydrolases are also commonly referred to as “theCel6 family.” In some embodiments, the CBH2 is functionally linked to acarbohydrate binding module (CBM), such as a Family 1 cellulose bindingdomain.

As used herein, the terms “beta-glucosidase,” “cellobiase,” and “BGL”refers to a category of cellulases (EC 3.2.1.21) that catalyze thehydrolysis of cellobiose to glucose. The term “beta-glucosidase” isfurther defined herein as a beta-D-glucoside glucohydrolase (E.C.3.2.1.21), which catalyzes the hydrolysis of terminal non-reducingbeta-D-glucose residues with the release of beta-D-glucose.Beta-glucosidase activity can be determined using any suitable method(See e.g., J. Basic Microbiol., 42: 55-66 [2002]). One unit ofbeta-glucosidase activity is defined as 1.0 pmole of p-nitrophenolproduced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodiumcitrate containing 0.01% TWEEN® 20.

As used herein, the term “glycoside hydrolase 61” and “GH61” refers to acategory of cellulases that enhance cellulose hydrolysis when used inconjunction with one or more additional cellulases. The GH61 family ofcellulases is described, for example, in the Carbohydrate Active Enzymes(CAZY) database (See e.g., Harris et al., Biochem., 49(15):3305-16[2010]).

A “hemicellulase” as used herein, refers to a polypeptide that cancatalyze hydrolysis of hemicellulose into small polysaccharides such asoligosaccharides, or monomeric saccharides. Hemicellulloses includexylan, glucuonoxylan, arabinoxylan, glucomannan and xyloglucan.Hemicellulases include, for example, the following: endoxylanases,beta-xylosidases, alpha-L-arabinofuranosidases, alpha-D-glucuronidases,feruloyl esterases, coumaroyl esterases, alpha-galactosidases,beta-galactosidases, beta-mannanases, and beta-mannosidases.

As used herein, the terms “xylan degrading activity” and “xylanolyticactivity” are defined herein as a biological activity that hydrolyzesxylan-containing material. The two basic approaches for measuringxylanolytic activity include: (1) measuring the total xylanolyticactivity, and (2) measuring the individual xylanolytic activities(endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases) (See e.g., Biely and Puchard, J. Sci. FoodAgr. 86:1636-1647 [2006]; Spanikova and Biely, FEBS Lett., 580:4597-4601[2006]; and Herrmann et al., Biochem. J., 321:375-381 [1997]).

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including oat spelt,beechwood, and larchwood xylans, or by photometric determination of dyedxylan fragments released from various covalently dyed xylans. A commontotal xylanolytic activity assay is based on production of reducingsugars from polymeric 4-O-methyl glucuronoxylan (See e.g., Bailey etal., J. Biotechnol., 23:257-270 [1992]). In some embodiments, xylandegrading activity is determined by measuring the increase in hydrolysisof birchwood xylan (Sigma Chemical Co., Inc., St. Louis, Mo., USA) byxylan-degrading enzyme(s) under the following typical conditions: 1 mLreactions, 5 mg/mL substrate (total solids), 5 mg of xylanolyticprotein/g of substrate, 50 mM sodium acetate pH 5, 50° C., 24 hours,sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH) assay (Seee.g., Lever, Anal. Biochem., 47:273-279 [1972]).

As used herein the term “xylanase activity” refers to a1,4-beta-D-xylan-xylohydrolase activity (E.C. 3.2.1.8) that catalyzesthe endo-hydrolysis of 1,4-beta-D-xylosidic linkages in xylans. In someembodiments, xylanase activity is determined using birchwood xylan assubstrate. One unit of xylanase activity is defined as 1.0 μmole ofreducing sugar (measured in glucose equivalents; See e.g., Lever, Anal.Biochem., 47:273-279 [1972]) produced per minute during the initialperiod of hydrolysis at 50° C., pH 5 from 2 g of birchwood xylan perliter as substrate in 50 mM sodium acetate containing 0.01% TWEEN® 20.

As used herein, the term “beta-xylosidase activity” refers to abeta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes theexo-hydrolysis of short beta (1→4)-xylooligosaccharides, to removesuccessive D-xylose residues from the non-reducing termini. In someembodiments of the present invention, one unit of beta-xylosidaseactivity is defined as 1.0 μmole of p-nitrophenol produced per minute at40° C., pH 5 from 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100mM sodium citrate containing 0.01% TWEEN® 20.

As used herein, the term “acetylxylan esterase activity” refers to acarboxylesterase activity (EC 3.1.1.72) that catalyses the hydrolysis ofacetyl groups from polymeric xylan, acetylated xylose, acetylatedglucose, alpha-napthyl acetate, and p-nitrophenyl acetate. In someembodiments of the present invention, acetylxylan esterase activity isdetermined using 0.5 mM p-nitrophenylacetate as substrate in 50 mMsodium acetate pH 5.0 containing 0.01% TWEEN® 20. One unit ofacetylxylan esterase activity is defined as the amount of enzyme capableof releasing 1 pmole of p-nitrophenolate anion per minute at pH 5, 25°C.

As used herein, the term “feruloyl esterase activity” refers to a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase activity (EC 3.1.1.73) thatcatalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl(feruloyl)group from an esterified sugar, which is usually arabinose in “natural”substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloylesterase is also known as ferulic acid esterase, hydroxycinnamoylesterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, orFAE-II. In some embodiments of the present invention, feruloyl esteraseactivity is determined using 0.5 mM p-nitrophenylferulate as substratein 50 mM sodium acetate pH 5.0. One unit of feruloyl esterase activityequals the amount of enzyme capable of releasing 1 μmole ofp-nitrophenolate anion per minute at pH 5, 25° C.

As used herein, the term “alpha-glucuronidase activity” refers to analpha-D-glucosiduronate glucuronohydrolase activity (EC 3.2.1.139) thatcatalyzes the hydrolysis of an alpha-D-glucuronoside to D-glucuronateand an alcohol. One unit of alpha-glucuronidase activity equals theamount of enzyme capable of releasing 1 pmole of glucuronic or4-O-methylglucuronic acid per minute at pH 5, 40° C. (See e.g., deVries, J. Bacteriol., 180:243-249 [1998]).

As used herein the term “alpha-L-arabinofuranosidase activity” refers toan alpha-L-arabinofuranoside arabinofuranohydrolase activity (EC3.2.1.55) that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeactivity acts on alpha-L-arabinofuranosides, alpha-L-arabinanscontaining (1,3)- and/or (1,5)-linkages, arabinoxylans, andarabinogalactans. Alpha-L-arabinofuranosidase is also known asarabinosidase, alpha-arabinosidase, alpha-L-arabinosidase,alpha-arabinofuranosidase, arabinofuranosidase, polysaccharidealpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase,L-arabinosidase and alpha-L-arabinanase. For purposes of the presentinvention, alpha-L-arabinofuranosidase activity is determined using 5 mgof medium viscosity wheat arabinoxylan (Megazyme International Ireland,Ltd., Bray, Co. Wicklow, Ireland) per mL of 100 mM sodium acetate pH 5in a total volume of 200 μL for 30 minutes at 40° C. followed byarabinose analysis by AMINEX®. HPX-87H column chromatography (Bio-RadLaboratories, Inc., Hercules, Calif., USA).

Enzymatic lignin depolymerization can be accomplished by ligninperoxidases, manganese peroxidases, laccases and cellobiosedehydrogenases (CDH), often working in synergy. These extracellularenzymes, essential for lignin degradation, are often referred to as“lignin-modifying enzymes” or “LMEs.” Three of these enzymes comprisetwo glycosylated heme-containing peroxidases: lignin peroxidase (LIP);Mn-dependent peroxidase (MNP); and, a copper-containing phenoloxidaselaccase (LCC). Although the details of the reaction scheme of ligninbiodegradation are not fully understood to date, without being bound bytheory, it is suggested that these enzymes employ free radicals fordepolymerization reactions.

As used herein, the term “laccase” refers to the copper containingoxidase enzymes that are found in many plants, fungi and microorganisms.Laccases are enzymatically active on phenols and similar molecules andperform a one electron oxidation. Laccases can be polymeric and theenzymatically active form can be a dimer or trimer.

As used herein, the term “Mn-dependent peroxidase” refers to peroxidasesthat require Mn. The enzymatic activity of Mn-dependent peroxidase (MnP)in is dependent on Mn²⁺. Without being bound by theory, it has beensuggested that the main role of this enzyme is to oxidize Mn²⁺ to Mn³⁺(See e.g., Glenn et al. Arch. Biochem. Biophys., 251:688-696 [1986]).Subsequently, phenolic substrates are oxidized by the Mn³⁺ generated.

As used herein, the term “lignin peroxidase” refers to an extracellularheme that catalyses the oxidative depolymerization of dilute solutionsof polymeric lignin in vitro. Some of the substrates of LiP, mostnotably 3,4-dimethoxybenzyl alcohol (veratryl alcohol, VA), are activeredox compounds that have been shown to act as redox mediators. VA is asecondary metabolite produced at the same time as LiP by ligninolyticcultures of P. chrysosporium and without being bound by theory, has beenproposed to function as a physiological redox mediator in theLiP-catalysed oxidation of lignin in vivo (See e.g., Harvey et al., FEBSLett. 195:242-246 [1986]).

As used herein, the term “glucoamylase” (EC 3.2.1.3) refers to enzymesthat catalyze the release of D-glucose from non-reducing ends of oligo-and poly-saccharide molecules. Glucoamylase is also generally considereda type of amylase known as amylo-gludosidase.

As used herein, the term “amylase” (EC 3.2.1.1) refers to starchcleaving enzymes that degrade starch and related compounds byhydrolyzing the alpha-1,4 and/or alpha-1,6 glucosidic linkages in anendo- or an exo-acting fashion. Amylases include alpha-amylases (EC3.2.1.1); beta-amylases (3.2.1.2), amylo-amylases (EC 3.2.1.3),alpha-glucosidases (EC 3.2.1.20), pullulanases (EC 3.2.1.41), andisoamylases (EC 3.2.1.68). In some embodiments, the amylase is analpha-amylase.

As used herein, the term “pectinase” refers to enzymes that catalyze thehydrolysis of pectin into smaller units such as oligosaccharide ormonomeric saccharides. In some embodiments, the enzyme mixtures compriseany pectinase, for example an endo-polygalacturonase, a pectin methylesterase, an endo-galactanase, a pectin acetyl esterase, an endo-pectinlyase, pectate lyase, alpha rhamnosidase, an exo-galacturonase, anexo-polygalacturonate lyase, a rhamnogalacturonan hydrolase, arhamnogalacturonan lyase, a rhamnogalacturonan acetyl esterase, arhamnogalacturonan galacturonohydrolase and/or a xylogalacturonase.

As used herein, the term “endo-polygalacturonase” (EC 3.2.1.15) refersto enzymes that catalyze the random hydrolysis of1,4-alpha-D-galactosiduronic linkages in pectate and othergalacturonans. This enzyme may also be referred to as “polygalacturonasepectin depolymerase,” “pectinase,” “endopolygalacturonase,” “pectolase,”“pectin hydrolase,” “pectin polygalacturonase,”“poly-alpha-1,4-galacturonide glycanohydrolase,” “endogalacturonase,”“endo-D-galacturonase,” or “poly(1,4-alpha-D-galacturonide)glycanohydrolase.”

As used herein, the term “pectin methyl esterase” (EC 3.1.1.11) refersto enzymes that catalyze the reaction: pectin+n H₂O=n methanol+pectate.The enzyme may also been known as “pectin esterase,” “pectindemethoxylase,” “pectin methoxylase,” “pectin methylesterase,”“pectase,” “pectinoesterase,” or “pectin pectylhydrolase.”

As used herein, the term “endo-galactanase” (EC 3.2.1.89) refers toenzymes that catalyze the endohydrolysis of 1,4-beta-D-galactosidiclinkages in arabinogalactans. The enzyme may also be known as“arabinogalactan endo-1,4-beta-galactosidase,”“endo-1,4-beta-galactanase,” galactanase,” “arabinogalactanase,” or“arabinogalactan 4-β-D-galactanohydrolase.”

As used herein, the term “pectin acetyl esterase” refers to enzymes thatcatalyze the deacetylation of the acetyl groups at the hydroxyl groupsof GaIUA residues of pectin.

As used herein, the term “one endo-pectin lyase” (EC 4.2.2.10) refers toenzymes that catalyze the eliminative cleavage of(1→4)-alpha-D-galacturonan methyl ester to give oligosaccharides with4-deoxy-6-O-methyl-alpha-D-galact-4-enuronosyl groups at theirnon-reducing ends. The enzyme may also be known as “pectin lyase,”“pectin transeliminase.” “endo-pectin lyase,” “polymethylgalacturonictranseliminase,” “pectin methyltranseliminase,” “pectolyase,” “PL,”“PNL,” “PMGL,” or “(1→4)-6-O-methyl-α-D-galacturonan lyase.”

As used herein, the term “pectate lyase” (EC 4.2.2.2) refers to enzymesthat catalyze the eliminative cleavage of (1→4)-alpha-D-galacturonan togive oligosaccharides with 4-deoxy-alpha-D-galact-4-enuronosyl groups attheir non-reducing ends. The enzyme may also be known as“polygalacturonic transeliminase,” “pectic acid transeliminase,”“polygalacturonate lyase,” “endopectin methyltranseliminase,” “pectatetranseliminase,” “endogalacturonate transeliminase,” “pectic acidlyase,” “pectic lyase,” “alpha-1,4-D-endopolygalacturonic acid lyase,”“PGA lyase,” “PPase-N,” “endo-alpha-1,4-polygalacturonic acid lyase,”“polygalacturonic acid lyase,” “pectin trans-eliminase,”“polygalacturonic acid trans-eliminase,” or “(1→4)-alpha-D-galacturonanlyase.”

As used herein, the term “alpha-rhamnosidase” (EC 3.2.1.40) refers toenzymes that catalyze the hydrolysis of terminal non-reducingalpha-L-rhamnose residues in alpha-L-rhamnosides or alternatively inrhamnogalacturonan. This enzyme may also be known as“alpha-L-rhamnosidase T,” “alpha-L-rhamnosidase N,” or“alpha-L-rhamnoside rhamnohydrolase.”

As used herein, the term “exo-galacturonase” (EC 3.2.1.82) refers toenzymes that hydrolyze pectic acid from the non-reducing end, releasingdigalacturonate. The enzyme may also be known as“exo-poly-alpha-galacturonosidase,” “exopolygalacturonosidase,” or“exopolygalacturanosidase.”

As used herein, the term “exo-galacturan 1,4-alpha galacturonidase” (EC3.2.1.67) refers to enzymes that catalyze reactions of the followingtypes:(1,4-alpha-D-galacturonide)n+H2O=(1,4-alpha-D-galacturonide)n-i+D-galacturonate.The enzyme may also be known as “poly[1->4)alpha-D-galacturonide]galacturonohydrolase,” “exopolygalacturonase,”“poly(galacturonate) hydrolase,” “exo-D-galacturonase,”“exo-D-galacturonanase,” “exopoly-D-galacturonase,” or“poly(1,4-alpha-D-galacturonide) galacturonohydrolase.”

As used herein, the term “exopolygalacturonate lyase” (EC 4.2.2.9)refers to enzymes that catalyze eliminative cleavage of4-(4-deoxy-α-D-galact-4-enuronosyl)-D-galacturonate from the reducingend of pectate (i.e. de-esterified pectin). This enzyme may be known as“pectate disaccharide-lyase,” “pectate exo-lyase,” “exopectic acidtranseliminase,” “exopectate lyase,” “exopolygalacturonicacid-trans-eliminase,” “PATE,” “exo-PATE,” “exo-PGL,” or“(1→4)-alpha-D-galacturonan reducing-end-disaccharide-lyase.”

As used herein, the term “rhamnogalacturonanase” refers to enzymes thathydrolyze the linkage between galactosyluronic acid and rhamnopyranosylin an endo-fashion in strictly alternating rhamnogalacturonanstructures, consisting of the disaccharide[(1,2-alpha-L-rhamnoyl-(1,4)-alpha-galactosyluronic acid].

As used herein, the term “rhamnogalacturonan lyase” refers to enzymesthat cleave alpha-L-Rhap-(1→4)-alpha-D-GalpA linkages in an endo-fashionin rhamnogalacturonan by beta-elimination.

As used herein, the term “rhamnogalacturonan acetyl esterase” refers toenzymes that catalyze the deacetylation of the backbone of alternatingrhamnose and galacturonic acid residues in rhamnogalacturonan.

As used herein, the term “rhamnogalacturonan galacturonohydrolase”refers to enzymes that hydrolyze galacturonic acid from the non-reducingend of strictly alternating rhamnogalacturonan structures in anexo-fashion. This enzyme may also be known as “xylogalacturonanhydrolase.”

As used herein, the term “endo-arabinanase” (EC 3.2.1.99) refers toenzymes that catalyze endohydrolysis of 1,5-alpha-arabinofuranosidiclinkages in 1,5-arabinans. The enzyme may also be known as“endo-arabinase,” “arabinan endo-1,5-α-L-arabinosidase,”“endo-1,5-alpha-L-arabinanase,” “endo-alpha-1,5-arabanase,”“endo-arabanase,” or “1,5-alpha-L-arabinan1,5-alpha-L-arabinanohydrolase.”

As used herein, “protease” includes enzymes that hydrolyze peptide bonds(peptidases), as well as enzymes that hydrolyze bonds between peptidesand other moieties, such as sugars (glycopeptidases). Many proteases arecharacterized under EC 3.4, and are suitable for use in the presentinvention. Some specific types of proteases include but are not limitedto, cysteine proteases including pepsin, papain and serine proteasesincluding chymotrypsins, carboxypeptidases and metalloendopeptidases.

As used herein, “lipase” includes enzymes that hydrolyze lipids, fattyacids, and acylglycerides, including phosphoglycerides, lipoproteins,diacylglycerols, and the like. In plants, lipids are used as structuralcomponents to limit water loss and pathogen infection. These lipidsinclude waxes derived from fatty acids, as well as cutin and suberin.

As used herein, the terms “isolated” and “purified” are used to refer toa molecule (e.g., an isolated nucleic acid, polypeptide [including, butnot limited to enzymes], etc.) or other component that is removed fromat least one other component with which it is naturally associated. Itis intended that the term encompass any suitable method for removing atleast one component with which the molecule is naturally associated. Insome embodiments, the terms also encompass cells that are separated fromother cells and/or media components. It is intended that any suitableseparation method finds use in the present invention.

As used herein, the term “purification process” used in reference to anenzyme mixture encompasses any process that physically removes anundesired component of the enzyme mixture. Thus, in some embodiments,purification processes provided herein include purificationmethodologies that physically remove one or more glucose and/orcellobiose oxidizing enzymes from the enzyme mixture or vice versa. Itis contemplated that any suitable purification process known in the artwill find use in the present invention. Indeed, it is not intended thatthe present invention be limited to any particular purification process.

As used herein, the term “cell-free enzyme mixture” comprises enzymesthat have been separated from any cells, including the cells thatsecreted the enzymes. Cell-free enzyme mixtures can be prepared by anyof a variety of methodologies that are known in the art, such asfiltration or centrifugation methodologies. In some embodiments, theenzyme mixture can be, for example, partially cell-free, substantiallycell-free, or entirely cell-free.

As used herein, “polynucleotide” refers to a polymer ofdeoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form, and complements thereof.

The terms “protein” and “polypeptide” are used interchangeably herein torefer to a polymer of amino acid residues.

In addition, the terms “amino acid” “polypeptide,” and “peptide”encompass naturally-occurring and synthetic amino acids, as well asamino acid analogs. Naturally occurring amino acids are those encoded bythe genetic code, as well as those amino acids that are later modified(e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine). As usedherein, the term “amino acid analogs” refers to compounds that have thesame basic chemical structure as a naturally occurring amino acid (i.e.,an α-carbon that is bound to a hydrogen, a carboxyl group, an aminogroup, and an R group, including but not limited to homoserine,norleucine, methionine sulfoxide, and methionine methyl sulfonium). Insome embodiments, these analogs have modified R groups (e.g.,norleucine) and/or modified peptide backbones, but retain the same basicchemical structure as a naturally occurring amino acid.

Amino acids are referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

An amino acid or nucleotide base “position” is denoted by a number thatsequentially identifies each amino acid (or nucleotide base) in thereference sequence based on its position relative to the N-terminus (or5′-end). Due to deletions, insertions, truncations, fusions, and thelike that must be taken into account when determining an optimalalignment, the amino acid residue number in a test sequence determinedby simply counting from the N-terminus will not necessarily be the sameas the number of its corresponding position in the reference sequence.For example, in a case where a test sequence has a deletion relative toan aligned reference sequence, there will be no amino acid in thevariant that corresponds to a position in the reference sequence at thesite of deletion. Where there is an insertion in an aligned testsequence, that insertion will not correspond to a numbered amino acidposition in the reference sequence. In the case of truncations orfusions there can be stretches of amino acids in either the reference oraligned sequence that do not correspond to any amino acid in thecorresponding sequence.

As used herein, the terms “numbered with reference to” or “correspondingto,” when used in the context of the numbering of a given amino acid orpolynucleotide sequence, refers to the numbering of the residues of aspecified reference sequence when the given amino acid or polynucleotidesequence is compared to the reference sequence.

As used herein, the term “reference enzyme” refers to an enzyme to whichanother enzyme of the present invention (e.g., a “test” enzyme) iscompared in order to determine the presence of an improved property inthe other enzyme being evaluated. In some embodiments, a referenceenzyme is a wild-type enzyme. In some embodiments, the reference enzymeis an enzyme to which a test enzyme of the present invention is comparedin order to determine the presence of an improved property in the testenzyme being evaluated, including but not limited to improvedthermoactivity, improved thermostability, and/or improved stability. Insome embodiments, a reference enzyme is a wild-type enzyme.

As used herein, the term “biologically active fragment,” refers to apolypeptide that has an amino-terminal and/or carboxy-terminaldeletion(s) and/or internal deletion(s), but where the remaining aminoacid sequence is identical to the corresponding positions in thesequence to which it is being compared and that retains substantiallyall of the activity of the full-length polypeptide.

As used herein, the term “recombinant” refers to a polynucleotide orpolypeptide that does not naturally occur in a host cell. In someembodiments, recombinant molecules contain two or morenaturally-occurring sequences that are linked together in a way thatdoes not occur naturally. In some embodiments, “recombinant cells”express genes that are not found in identical form within the native(i.e., non-recombinant) form of the cell and/or express native genesthat are otherwise abnormally over-expressed, under-expressed, and/ornot expressed at all due to deliberate human intervention. Recombinantcells contain at least one recombinant polynucleotide or polypeptide. Anucleic acid construct, nucleic acid (e.g., a polynucleotide),polypeptide, or host cell is referred to herein as “recombinant” when itis non-naturally occurring, artificial or engineered. “Recombination,”“recombining” and generating a “recombined” nucleic acid generallyencompass the assembly of at least two nucleic acid fragments.

The present invention also provides a recombinant nucleic acid constructcomprising at least one CDH polynucleotide sequence that hybridizesunder stringent hybridization conditions to the complement of apolynucleotide which encodes a polypeptide having the amino acidsequence of SEQ ID NOS:6 and/or 8.

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well-characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. As used herein, the term “stringenthybridization wash conditions” in the context of nucleic acidhybridization experiments, such as Southern and Northern hybridizations,are sequence dependent, and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in Tijssen, 1993, “Laboratory Techniques in Biochemistry andMolecular Biology-Hybridization with Nucleic Acid Probes,” Part I,Chapter 2 (Elsevier, New York), which is incorporated herein byreference. For polynucleotides of at least 100 nucleotides in length,low to very high stringency conditions are defined as follows:prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200μg/ml sheared and denatured salmon sperm DNA, and either 25% formamidefor low stringencies, 35% formamide for medium and medium-highstringencies, or 50% formamide for high and very high stringencies,following standard Southern blotting procedures. For polynucleotides ofat least 100 nucleotides in length, the carrier material is finallywashed three times each for 15 minutes using 2×SSC, 0.2% SDS 50° C. (lowstringency), at 55° C. (medium stringency), at 60° C. (medium-highstringency), at 65° C. (high stringency), or at 70° C. (very highstringency).

Moderately stringent conditions encompass those known in the art anddescribed in various standard texts and include the use of washingsolution and hybridization conditions (e.g., temperature, ionic strengthand % SDS). An example of moderately stringent conditions involvesovernight incubation at 37° C. in a solution comprising: 20% formamide,5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denaturedsheared salmon sperm DNA, followed by washing the filters in 1×SSC atabout 37-50° C. The skilled artisan will recognize how to adjust thetemperature, ionic strength, etc. as necessary to accommodate factorssuch as probe length and the like.

As used in some embodiments herein, stringent conditions or highstringency conditions utilize: (1) low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) 50% formamide,5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmonsperm DNA (50 μg/mL), 0.1% SDS, and 10% dextran sulfate at 42° C., withwashes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50%formamide at 55° C., followed by a high-stringency wash consisting of0.1×SSC containing EDTA at 55° C.

As used herein, “similarity” refers to an identical or conservativeamino acid substitution thereof as defined below. Accordingly, a changeto an identical or conservative substitution for the purposes ofsimilarity is viewed as not comprising a change. A deletion of an aminoacid or a non-conservative amino acid substitution is viewed herein ascomprising a change. Calculation of percent similarity is performed inthe same manner as performed for percent identity. A conservative aminoacid substitution can be a substitution such as the conservativesubstitutions shown in Table A. The substitutions shown are based onamino acid physical-chemical properties, and as such, are independent oforganism. In some embodiments, the conservative amino acid substitutionis a substitution listed under the heading of exemplary substitutions.

TABLE A Substitutions Original Residue Conservative SubstitutionsExemplary Substitutions Ala (A) val; leu; ile Val Arg (R) lys; gln; asnLys Asn (N) gln; his; lys; arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln(Q) Asn Asn Glu (E) Asp Asp Gly (G) pro; ala Ala His (H) asn; gln; lys;arg Arg Ile (I) leu; val; met; ala; phe Leu Leu (L) ile; val; met; ala;phe Ile Lys (K) arg; gln; asn Arg Met (M) leu; phe; ile Leu Phe (F) leu;val; ile; ala; tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser SerTrp (W) tyr; phe Tyr Tyr (Y) trp; phe; thr; ser Phe Val (V) ile; leu;met; phe; ala Leu

As used herein, “identity” and “percent identity,” in the context of twoor more polypeptide sequences, refers to two or more sequences orsubsequences that are the same or have a specified percentage of aminoacid residues that are the same (e.g., share at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about88% identity, at least about 89%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity) over a specified region to areference sequence, when compared and aligned for maximum correspondenceover a comparison window, or designated region as measured using asequence comparison algorithms or by manual alignment and visualinspection.

In some embodiments, the terms “percent identity,” “% identity”,“percent identical,” and “% identical,” are used interchangeably hereinto refer to the percent amino acid or polynucleotide sequence identitythat is obtained by ClustalW analysis (version W 1.8 available fromEuropean Bioinformatics Institute, Cambridge, UK), counting the numberof identical matches in the alignment and dividing such number ofidentical matches by the length of the reference sequence, and using thefollowing ClustalW parameters to achieve slow/more accurate pairwiseoptimal alignments—DNA/Protein Gap Open Penalty: 15/10; DNA/Protein GapExtension Penalty: 6.66/0.1; Protein weight matrix: Gonnet series; DNAweight matrix: Identity.

Two sequences are “aligned” when they are aligned for similarity scoringusing a defined amino acid substitution matrix (e.g., BLOSUM62), gapexistence penalty and gap extension penalty so as to arrive at thehighest score possible for that pair of sequences. Amino acidsubstitution matrices and their use in quantifying the similaritybetween two sequences are well known in the art (See, e.g., Dayhoff etal., in Dayhoff [ed.], Atlas of Protein Sequence and Structure,” Vol. 5,Suppl. 3, Natl. Biomed. Res. Round., Washington D.C. [1978]; pp.345-352; and Henikoff et al., Proc. Natl. Acad. Sci. USA, 89:10915-10919[1992], both of which are incorporated herein by reference). TheBLOSUM62 matrix is often used as a default scoring substitution matrixin sequence alignment protocols such as Gapped BLAST 2.0. The gapexistence penalty is imposed for the introduction of a single amino acidgap in one of the aligned sequences, and the gap extension penalty isimposed for each additional empty amino acid position inserted into analready opened gap. The alignment is defined by the amino acid positionof each sequence at which the alignment begins and ends, and optionallyby the insertion of a gap or multiple gaps in one or both sequences soas to arrive at the highest possible score. While optimal alignment andscoring can be accomplished manually, the process is facilitated by theuse of a computer-implemented alignment algorithm (e.g., gapped BLAST2.0; See, Altschul et al., Nucleic Acids Res., 25:3389-3402 [1997],which is incorporated herein by reference), and made available to thepublic at the National Center for Biotechnology Information Website).Optimal alignments, including multiple alignments can be prepared usingreadily available programs such as PSI-BLAST (See e.g., Altschul et al.,supra).

The present invention also provides a recombinant nucleic acid constructcomprising a CDH polynucleotide sequence that hybridizes under stringenthybridization conditions to the complement of a polynucleotide whichencodes a polypeptide having the amino acid sequence of SEQ ID NO:6and/or 8. Two nucleic acid or polypeptide sequences that have 100%sequence identity are said to be “identical.” A nucleic acid orpolypeptide sequence is said to have “substantial sequence identity” toa reference sequence when the sequences have at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, or greater sequenceidentity as determined using the methods described herein, such as BLASTusing standard parameters.

As used herein, a “secretion signal peptide” can be a propeptide, aprepeptide or both. For example, the term “propeptide” refers to aprotein precursor that is cleaved to yield a “mature protein.” Thesignal peptide is cleaved from the pre-protein by a signal peptidaseprior to secretion to result in the “mature” or “secreted” protein. Theterms “prepeptide” ad “pre-protein” refer to a polypeptide synthesizedwith an N-terminal signal peptide that targets it for secretion.Accordingly, a “pre-pro-peptide” is a polypeptide that contains a signalpeptide that targets the polypeptide for secretion and which is cleavedoff to yield a mature polypeptide. Signal peptides are found at theN-terminus of the protein and are typically composed of between 6 to 136basic and hydrophobic amino acids.

As used herein, “transcription” and like terms refer to the conversionof the information encoded in a gene to an RNA transcript. Accordingly,a reduction of the transcription level of a glucose and/or cellobioseoxidizing enzyme is a reduction in the amount of RNA transcript of anRNA coding for a glucose and/or cellobiose oxidizing enzyme.

As used herein, a “vector” is a polynucleotide construct for introducinga polynucleotide sequence into a cell. In some embodiments, the vectorcomprises a suitable control sequence operably linked to and capable ofeffecting the expression of the polypeptide encoded in thepolynucleotide sequence in a suitable host. An “expression vector” has apromoter sequence operably linked to the polynucleotide sequence (e.g.,transgene) to drive expression in a host cell, and in some embodiments atranscription terminator sequence. In some embodiments, the vectors aredeletion vectors. In some embodiments, vectors comprise polynucleotidesequences that produce small interfering RNA or antisense RNAtranscripts that interfere with the translation of a targetpolynucleotide sequence.

As used herein, a “deletion vector” comprises polynucleotide sequenceshomologous to a polynucleotide sequences 5′ and 3′ to a target sequenceto be deleted from a host genome so as to direct recombination andreplacement of the target sequence with a polynucleotide between the 5′and 3′ targeting sequences.

As used herein, the term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation, andpost-translational modification. In some embodiments, the term alsoencompasses secretion of the polypeptide from a cell. In general theterm, “expression” refers to conversion of the information encoded in agene to the protein encoded by that gene. Thus, a “reduction of theamount of an expressed glucose and/or cellobiose oxidizing enzyme” is areduction in the amount of the glucose and/or cellobiose oxidizingenzyme that is eventually translated by the cell.

As used herein, the term “overexpress” is intended to encompassincreasing the expression of a protein to a level greater than the cellnormally produces. It is intended that the term encompass overexpressionof endogenous, as well as heterologous proteins. In some embodiments,overexpression includes an elevated transcription rate and/or level ofthe gene compared to the endogenous transcription rate and/or level forthat gene. For example, in some embodiments, a heterologous gene isintroduced into a fungal cell to express a gene encoding a heterologousenzyme such as a beta-glucosidase from another organism. In some otherembodiments, a heterologous gene is introduced into a fungal cell tooverexpress a gene encoding a homologous enzyme such as abeta-glucosidase.

In some embodiments, the heterologous gene is a gene that has beenmodified to overexpress the gene product. In some embodiments,“overexpression” refers to any state in which a gene is caused to beexpressed at an elevated rate or level as compared to the endogenousexpression rate or level for that gene. In some embodiments,overexpression includes elevated translation rate and/or level of thegene compared to the endogenous translation rate and/or level for thatgene. As used herein, the term “produces” refers to the production ofproteins and/or other compounds by cells. It is intended that the termencompass any step involved in the production of polypeptides including,but not limited to, transcription, post-transcriptional modification,translation, and post-translational modification. In some embodiments,the term also encompasses secretion of the polypeptide from a cell.

As used herein, a “polynucleotide sequence that has been adapted forexpression” is a polynucleotide sequence that has been inserted into anexpression vector or otherwise modified to contain regulatory elementsnecessary for expression of the polynucleotide in the host cell,positioned in such a manner as to permit expression of thepolynucleotide in the host cell. Such regulatory elements required forexpression include promoter sequences, transcription initiationsequences and, optionally, enhancer sequences. For example, in someembodiments, a polynucleotide sequence is inserted into a plasmid vectoradapted for expression in the fungal host cell.

As used herein, the term “operably linked” refers to a configuration inwhich a control sequence is appropriately placed at a position relativeto the coding sequence of the DNA sequence such that the controlsequence influences the expression of a polypeptide.

As used herein, an amino acid or nucleotide sequence (e.g., a promotersequence, signal peptide, terminator sequence, etc.) is “heterologous”to another sequence with which it is operably linked if the twosequences are not associated in nature.

As used herein, a “heterologous enzyme” refers to an enzyme that isencoded by a “heterologous gene.” However, it is also contemplated thata heterologous gene encodes an endogenous or homologous enzyme, asexplained below. In general, the term “heterologous gene” refers to agene that occurs in a form not found in a parental strain of the hostfungal cell (including but not limited to wild-type). Thus, in someembodiments, a heterologous gene is a gene that is derived from aspecies that is different from the species of the fungal cell expressingthe gene and recognized anamorphs, teleomorphs or taxonomic equivalentsof the fungal cell expressing the gene. In some embodiments, aheterologous gene is a modified version of a gene that is endogenous tothe host fungal cell, which endogenous gene has been subjected tomanipulation and then introduced or transformed into the host cell. Forexample, in some embodiments, a heterologous gene has an endogenouscoding sequence, but has modifications to the promoter sequence.Similarly, in some embodiments, a heterologous gene encodes the sameamino acid sequence as an endogenous gene, but has modifications to thecodon usage or to noncoding regions such as introns, or a combinationthereof. For example, in some embodiments, a heterologous gene comprisesmodifications to the coding sequence to encode a non-wild typepolypeptide. In some other embodiments, a heterologous gene has the samepromoter sequence, 5′ and 3′ untranslated regions and coding regions asa parental strain, but be located in another region of the samechromosome, or on an entirely different chromosome as compared to aparental strain of the host cell.

As used herein, an “endogenous” or “homologous” gene refers to a genethat is found in a parental strain of the host fungal cell (including,but not limited to wild-type).

As used herein, the term “introduced” used in the context of inserting anucleic acid sequence into a cell, means transformation, transduction,conjugation, transfection, and/or any other suitable method(s) known inthe art for inserting nucleic acid sequences into host cells. Anysuitable means for the introduction of nucleic acid into host cells finduse in the present invention.

As used herein, the terms “transformed” and “transformation” used inreference to a cell refer to a cell that has a non-native nucleic acidsequence integrated into its genome or has an episomal plasmid that ismaintained through multiple generations.

As used herein, the terms “host cell” and “host strain” refer tosuitable hosts for expression vectors comprising polynucleotidesequences (e.g., DNA) as provided herein. In some embodiments, the hostcells are prokaryotic or eukaryotic cells that have been transformed ortransfected with vectors constructed using recombinant techniques asknown in the art. Transformed hosts are capable of either replicatingvectors encoding at least one protein of interest and/or expressing thedesired protein of interest. In addition, reference to a cell of aparticular strain refers to a parental cell of the strain as well asprogeny and genetically modified derivatives. Genetically modifiedderivatives of a parental cell include progeny cells that contain amodified genome or episomal plasmids that confer for example, antibioticresistance, improved fermentation, etc. In some embodiments, host cellsare genetically modified to have characteristics that improve proteinsecretion, protein stability or other properties desirable forexpression and/or secretion of a protein. For example, knockout of Alp1function results in a cell that is protease deficient. Knockout of pyr5function results in a cell with a pyrimidine deficient phenotype. Insome embodiments, host cells are modified to delete endogenous cellulaseprotein-encoding sequences or otherwise eliminate expression of one ormore endogenous cellulases. In some embodiments, expression of one ormore endogenous cellulases is inhibited to increase production ofcellulases of interest. Genetic modification can be achieved by anysuitable genetic engineering techniques and/or classical microbiologicaltechniques (e.g., chemical or UV mutagenesis and subsequent selection).Using recombinant technology, nucleic acid molecules can be introduced,deleted, inhibited or modified, in a manner that results in increasedyields of enzyme within the organism or in the culture. For example,knockout of Alp1 function results in a cell that is protease deficient.Knockout of pyr5 function results in a cell with a pyrimidine deficientphenotype. In some genetic engineering approaches, homologousrecombination is used to induce targeted gene modifications byspecifically targeting a gene in vivo to suppress expression of theencoded protein. In an alternative approach, siRNA, antisense, and/orribozyme technology finds use in inhibiting gene expression.

As used herein, “gene deletion” and “deletion mutation” refer to amutation in which part of a gene is missing. Thus, a deletion is a lossor replacement of genetic material resulting in a complete or partialdisruption of the sequence of the DNA making up the gene. Any number ofnucleotides can be deleted, from a single base to an entire piece of achromosome. In some embodiments, complete or near-complete deletion ofthe gene sequence is contemplated. However, a deletion mutation need notcompletely remove the entire gene sequence for the glucose and/orcellobiose oxidizing enzyme in order to reduce the endogenous glucoseand/or cellobiose oxidizing enzyme activity secreted by the fungal cell.For example, a partial deletion that removes one or more nucleotidesencoding an amino acid in a glucose and/or cellobiose oxidizing enzymeactive site, encoding a secretion signal, or encoding another portion ofthe glucose and/or cellobiose oxidizing enzyme that plays a role inendogenous glucose and/or cellobiose oxidizing enzyme activity beingsecreted by the fungal cell.

As used herein, a “conditional mutation” is a mutation that haswild-type phenotype under certain environmental conditions and a mutantphenotype under certain other conditions.

As used herein, the terms “amplification” and “gene amplification” referto a method by which specific DNA sequences are disproportionatelyreplicated such that the amplified gene becomes present in a higher copynumber than was initially present in the genome. In some embodiments,selection of cells by growth in the presence of a drug (e.g., aninhibitor of an inhabitable enzyme) results in the amplification ofeither the endogenous gene encoding the gene product required for growthin the presence of the drug or by amplification of exogenous (i.e.,input) sequences encoding this gene product, or both. “Amplification” isa special case of nucleic acid replication involving templatespecificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, that is capable of acting as a synthesis initiation pointwhen placed under conditions in which synthesis of a primer extensionproduct which is complementary to a nucleic acid strand is induced(i.e., in the presence of nucleotides and an inducing agent such as DNApolymerase and at a suitable temperature and pH). The primer ispreferably single stranded for maximum efficiency in amplification, butmay alternatively be double stranded. If double stranded, the primer isfirst treated to separate its strands before being used to prepareextension products. In some embodiments, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. As known in the art, the exact lengths of the primers will dependon many factors, including temperature, source of primer and the use ofthe method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, that is capable of hybridizing to another oligonucleotideof interest. A probe may be single-stranded or double-stranded. Probesare useful in the detection, identification and isolation of particulargene sequences. It is contemplated that any probe used in the presentinvention will be labeled with any “reporter molecule,” so that isdetectable in any detection system, including, but not limited to enzyme(e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is not intendedthat the present invention be limited to any particular detection systemor label.

As used herein, the term “target,” when used in reference to thepolymerase chain reaction, refers to the region of nucleic acid boundedby the primers used for polymerase chain reaction. Thus, the “target” issought to be sorted out from other nucleic acid sequences. A “segment”is defined as a region of nucleic acid within the target sequence.

As used herein, the term “polymerase chain reaction” (PCR) refers to themethods of U.S. Pat. Nos. 4,683,195 4,683,202, and 4,965,188, herebyincorporated by reference, which include methods for increasing theconcentration of a segment of a target sequence in a mixture of genomicDNA without cloning or purification. This method for amplifying thetarget sequence is well known in the art.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A “restriction site” refers to a nucleotide sequence recognized andcleaved by a given restriction endonuclease and is frequently the sitefor insertion of DNA fragments. In some embodiments of the invention,restriction sites are engineered into the selective marker and into 5′and 3′ ends of the DNA construct.

As used herein, “homologous recombination” means the exchange of DNAfragments between two DNA molecules or paired chromosomes at the site ofidentical or nearly identical nucleotide sequences. In some embodiments,chromosomal integration is homologous recombination.

As used herein, the term “C1” refers to Myceliophthora thermophilia,including the fungal strain described by Garg (See, Garg, Mycopathol.,30: 3-4 [1966]). As used herein, “Chrysosporium lucknowense” includesthe strains described in U.S. Pat. Nos. 6,015,707, 5,811,381 and6,573,086; US Pat. Pub. Nos. 2007/0238155, US 2008/0194005, US2009/0099079; International Pat. Pub. Nos., WO 2008/073914 and WO98/15633, all of which are incorporated herein by reference, andinclude, without limitation, Chrysosporium lucknowense Garg 27K, VKM-F3500 D (Accession No. VKM F-3500-D), C1 strain UV13-6 (Accession No. VKMF-3632 D), C1 strain NG7C-19 (Accession No. VKM F-3633 D), and C1 strainUV18-25 (VKM F-3631 D), all of which have been deposited at theAll-Russian Collection of Microorganisms of Russian Academy of Sciences(VKM), Bakhurhina St. 8, Moscow, Russia, 113184, and any derivativesthereof. Although initially described as Chrysosporium lucknowense, C1may currently be considered a strain of Myceliophthora thermophila.Other C1 strains include cells deposited under accession numbers ATCC44006, CBS (Centraalbureau voor Schimmelcultures) 122188, CBS 251.72,CBS 143.77, CBS 272.77, CBS122190, CBS122189, and VKM F-3500D. ExemplaryC1 derivatives include modified organisms in which one or moreendogenous genes or sequences have been deleted or modified and/or oneor more heterologous genes or sequences have been introduced.Derivatives include, but are not limited to UV18#100f Δalp1, UV18#100fΔpyr5 Δalp1, UV18#100.f Δalp1 Δpep4 Δalp2, UV18#100.f Δpyr5 Δalp1 Δpep4Δalp2 and UV18#100.f Δpyr4 Δpyr5 Δalp1 Δpep4 Δalp2, as described in WO2008073914 and WO 2010107303, each of which is incorporated herein byreference.

As used herein, a “genetically modified” and/or “genetically engineeredcell” (e.g., a “genetically engineered fungal cell” and/or a“genetically modified fungal cell”) is a cell whose genetic material hasbeen altered using genetic engineering techniques. A geneticallymodified cell also refers to a derivative of or the progeny of a cellwhose genetic material has been altered using genetic engineeringtechniques. An example of a genetic modification as a result of geneticengineering techniques includes a modification to the genomic DNA;another example of a genetic modification as a result of geneticengineering techniques includes introduction of a stable heterologousnucleic acid into the cell. For example, as provided herein, agenetically modified fungal cell as provided herein is a fungal cellthat whose genetic material has been altered in such a way as to eitherreduce the amount of secreted glucose and/or cellobiose oxidizing enzymeactivity, or to reduce the ability of the secreted enzyme to oxidizecellobiose or glucose.

As used herein, the term “culturing” refers to growing a population ofmicrobial cells under suitable conditions in a liquid or solid medium.It is contemplated that the culturing be carried out in any suitableformat, equipment (e.g., shake flasks, fermentation tanks, bioreactors,etc.). It is also intended that the culturing be conducted using anysuitable process methods, including but not limited to batch, fed-batch,and/or continuous culturing. Indeed, it is contemplated that anycombination of suitable methods will find use.

In a “batch process,” all the necessary materials, with the exception ofoxygen for aerobic processes, are placed in a reactor at the start ofthe operation and the fermentation is allowed to proceed untilcompletion, at which point the product is harvested. In someembodiments, batch processes for producing the fungal cells, enzymes,and/or enzyme mixtures of the present invention are carried out in ashake-flask or a bioreactor.

In a “fed-batch process,” the culture is fed continuously orsequentially with one or more media components without the removal ofthe culture fluid.

In a “continuous process,” fresh medium is supplied and culture fluid isremoved continuously at volumetrically equal rates to maintain theculture at a steady growth rate. In reference to continuous processes,“steady state” refers to a state in which the concentration of reactantsdoes not vary appreciably, and “quasi-steady state” refers to a state inwhich, subsequent to the initiation of the reaction, the concentrationof reactants fluctuates within a range consistent with normal operationof the continuous hydrolysis process.

As used herein, the term “saccharification” refers to the process inwhich substrates (e.g., cellulosic biomass) are broken down via theaction of cellulases to produce fermentable sugars (e.g. monosaccharidessuch as but not limited to glucose).

As used herein, the term “fermentable sugars” refers to simple sugars(e.g., monosaccharides, disaccharides and short oligosaccharides),including but not limited to glucose, xylose, galactose, arabinose,mannose and sucrose. Indeed, a fermentable sugar is any sugar that amicroorganism can utilize or ferment.

As used herein the term “soluble sugars” refers to water-soluble pentoseand hexose monomers and oligomers of up to about six monomer units. Itis intended that the term encompass any water soluble mono- and/oroligosaccharides.

As used herein, the term “fermentation” is used broadly to refer to theprocess of obtaining energy from the oxidation of organic compounds(e.g., carbohydrates). Indeed, “fermentation” broadly refers to thechemical conversion of a sugar source to an end product through the useof a fermenting organism. In some embodiments, the term encompassescultivation of a microorganism or a culture of microorganisms that usesugars, such as fermentable sugars, as an energy source to obtain adesired product.

As used herein, the term “fermenting organism” refers to any organism,including prokaryotic, as well as eukaryotic organisms (e.g., bacterialorganisms, as well as fungal organisms such as yeast and filamentousfungi), suitable for producing a desired end product. Especiallysuitable fermenting organisms are able to ferment (i.e., convert)sugars, including but not limited to glucose, fructose, maltose, xylose,mannose and/or arabinose, directly or indirectly into at least onedesired end product. In some embodiments, yeast that find use in thepresent invention include, but are not limited to strains of the genusSaccharomyces (e.g., strains of Saccharomyces cerevisiae andSaccharomyces uvarum), strains of the genus Pichia (e.g., Pichiastipitis such as Pichia stipitis CBS 5773 and Pichia pastoris), andstrains of the genus Candida (e.g., Candida utilis, Candidaarabinofermentans, Candida diddensii, Candida sonorensis, Candidashehatae, Candida tropicalis, and Candida boidinii). Other fermentingorganisms include, but are not limited to strains of Zymomonas,Hansenula (e.g., Hansenula polymorphs and Hansenula anomala),Kluyveromyces (e.g., Kluyveromyces fragilis), and Schizosaccharomyces(e.g., Schizosaccharomyces pombe).

As used herein, the term “slurry” refers to an aqueous solution in whichare dispersed one or more solid components, such as a cellulosicsubstrate. Thus, the term “slurry” refers to a suspension of solids in aliquid. In some embodiments, the cellulosic substrate is slurried in aliquid at a concentration that is thick, but can still be pumped. Forexample, in some embodiments, the liquid is water, a recycled processstream, and/or a treated effluent. However, it is not intended that thepresent invention be limited to any particular liquid and/or solid.

As used herein, “cellulose” refers to a polymer of the simple sugarglucose linked by beta-1,4 glycosidic bonds.

As used herein, “cellobiose” refers to a water-soluble beta-1,4-linkeddimer of glucose.

The terms “biomass,” and “biomass substrate,” encompass any suitablematerials for use in saccharification reactions. The terms encompass,but are not limited to, materials that comprise cellulose (i.e.,“cellulosic biomass,” “cellulosic feedstock,” and “cellulosicsubstrate”), as well as lignocellulosic biomass. Indeed, the term“biomass” encompasses any living or dead biological material thatcontains a polysaccharide substrate, including but not limited tocellulose, starch, other forms of long-chain carbohydrate polymers, andmixtures of such sources. In some embodiments, it is assembled entirelyor primarily from glucose or xylose, and in some embodiments, optionallyalso contains various other pentose and/or hexose monomers. Biomass canbe derived from plants, animals, or microorganisms, and includes, but isnot limited to agricultural, industrial, and forestry residues,industrial and municipal wastes, and terrestrial and aquatic crops grownfor energy purposes. Examples of biomass substrates include, but are notlimited to, wood, wood pulp, paper pulp, corn fiber, corn grain, corncobs, crop residues such as corn husks, corn stover, grasses, wheat,wheat straw, barley, barley straw, hay, rice, rice straw, switchgrass,waste paper, paper and pulp processing waste, woody or herbaceousplants, fruit or vegetable pulp, distillers grain, grasses, rice hulls,cotton, hemp, flax, sisal, sugar cane bagasse, sorghum, soy,switchgrass, components obtained from milling of grains, trees,branches, roots, leaves, wood chips, sawdust, shrubs and bushes,vegetables, fruits, and flowers and any suitable mixtures thereof. Insome embodiments, the biomass comprises, but is not limited tocultivated crops (e.g., grasses, including C4 grasses, such as switchgrass, cord grass, rye grass, miscanthus, reed canary grass, or anycombination thereof), sugar processing residues, for example, but notlimited to, bagasse (e.g., sugar cane bagasse, beet pulp [e.g., sugarbeet], or a combination thereof), agricultural residues (e.g., soybeanstover, corn stover, corn fiber, rice straw, sugar cane straw, rice,rice hulls, barley straw, corn cobs, wheat straw, canola straw, oatstraw, oat hulls, corn fiber, hemp, flax, sisal, cotton, or anycombination thereof), fruit pulp, vegetable pulp, distillers' grains,forestry biomass (e.g., wood, wood pulp, paper pulp, recycled wood pulpfiber, sawdust, hardwood, such as aspen wood, softwood, or a combinationthereof). Furthermore, in some embodiments, the biomass comprisescellulosic waste material and/or forestry waste materials, including butnot limited to, paper and pulp processing waste, municipal paper waste,newsprint, cardboard and the like. In some embodiments, biomasscomprises one species of fiber, while in some alternative embodiments,the biomass comprises a mixture of fibers that originate from differentbiomasses. In some embodiments, the biomass also comprises transgenicplants that express ligninase and/or cellulase enzymes (See e.g., US2008/0104724 A1).

As used herein, “lignocellulose” refers to a matrix of cellulose,hemicellulose and lignin. Economic production of biofuels fromlignocellulosic biomass typically involves conversion of the celluloseand hemicellulose components to fermentable sugars, typicallymonosaccharides such as glucose (from the cellulose) and xylose andarabinose (from the hemicelluloses). Nearly complete conversion can beachieved by a chemical pretreatment of the lignocellulose followed byenzymatic hydrolysis with cellulase enzymes. The chemical pretreatmentstep renders the cellulose more susceptible to enzymatic hydrolysis and,in some cases, also hydrolyzes the hemicellulose component. Numerouschemical pretreatment processes are known in the art, and include, butare not limited to, mild acid pretreatment at high temperatures anddilute acid, ammonium pretreatment or organic solvent extraction.

Lignin is a more complex and heterogeneous biopolymer than eithercellulose or hemicellulose and comprises a variety of phenolic subunits.Enzymatic lignin depolymerization can be accomplished by ligninperoxidases, manganese peroxidases, laccases and cellobiosedehydrogenases (CDH), often working in synergy. However, as the namesuggests, CDH enzymes also oxidize cellobiose to cellobionolactone.Several reports indicate that the oxidation of cellobiose by CDHenhances the rate of cellulose hydrolysis by cellulases by virtue ofreducing the concentrations of cellobiose, which is a potent inhibitorof some cellulase components (Mansfield et al., Appl. Environ.Microbiol., 63: 3804-3809 [1997]; and Igarishi et al., Eur. J. Biochem.,253: 101-106 [1998]). Recently, it has been reported that CDHs canenhance the activity of cellulolytic enhancing proteins from GlycosylHydrolase family 61 (See e.g., WO2010/080532A1).

As used herein, the term “lignocellulosic biomass” refers to any plantbiomass comprising cellulose and hemicellulose, bound to lignin

In some embodiments, the biomass is optionally pretreated to increasethe susceptibility of cellulose to hydrolysis by chemical, physical andbiological pretreatments (such as steam explosion, pulping, grinding,acid hydrolysis, solvent exposure, and the like, as well as combinationsthereof). Various lignocellulosic feedstocks find use, including thosethat comprise fresh lignocellulosic feedstock, partially driedlignocellulosic feedstock, fully dried lignocellulosic feedstock, and/orany combination thereof. In some embodiments, lignocellulosic feedstockscomprise cellulose in an amount greater than about 20%, more preferablygreater than about 30%, more preferably greater than about 40% (w/w).For example, in some embodiments, the lignocellulosic material comprisesfrom about 20% to about 90% (w/w) cellulose, or any amount therebetween,although in some embodiments, the lignocellulosic material comprisesless than about 19%, less than about 18%, less than about 17%, less thanabout 16%, less than about 15%, less than about 14%, less than about13%, less than about 12%, less than about 11%, less than about 10%, lessthan about 9%, less than about 8%, less than about 7%, less than about6%, or less than about 5% cellulose (w/w). Furthermore, in someembodiments, the lignocellulosic feedstock comprises lignin in an amountgreater than about 10%, more typically in an amount greater than about15% (w/w). In some embodiments, the lignocellulosic feedstock comprisessmall amounts of sucrose, fructose and/or starch. The lignocellulosicfeedstock is generally first subjected to size reduction by methodsincluding, but not limited to, milling, grinding, agitation, shredding,compression/expansion, or other types of mechanical action. Sizereduction by mechanical action can be performed by any type of equipmentadapted for the purpose, for example, but not limited to, hammer mills,tub-grinders, roll presses, refiners and hydrapulpers. In someembodiments, at least 90% by weight of the particles produced from thesize reduction have lengths less than between about 1/16 and about 4 in(the measurement may be a volume or a weight average length). In someembodiments, the equipment used to reduce the particle size is a hammermill or shredder. Subsequent to size reduction, the feedstock istypically slurried in water, as this facilitates pumping of thefeedstock. In some embodiments, lignocellulosic feedstocks of particlesize less than about 6 inches do not require size reduction.

As used herein, the term “lignocellulosic feedstock” refers to any typeof lignocellulosic biomass that is suitable for use as feedstock insaccharification reactions.

As used herein, the term “pretreated lignocellulosic feedstock,” refersto lignocellulosic feedstocks that have been subjected to physicaland/or chemical processes to make the fiber more accessible and/orreceptive to the actions of cellulolytic enzymes, as described above.

As used herein, the terms “lignocellulose-competent,”“lignocellulose-utilizing” and like terms refer to an organism thatsecretes enzymes that participate in lignin breakdown and hydrolysis.For example, in some embodiments, lignocellulose-competent fungal cellssecrete one or more lignin peroxidases, manganese peroxidases, laccasesand/or cellobiose dehydrogenases (CDH). These extracellular enzymes,essential for lignin degradation, are often referred to as“lignin-modifying enzymes” or “LMEs.”

A biomass substrate is said to be “pretreated” when it has beenprocessed by some physical and/or chemical means to facilitatesaccharification. As described further herein, in some embodiments, thebiomass substrate is “pretreated,” or treated using methods known in theart, such as chemical pretreatment (e.g., ammonia pretreatment, diluteacid pretreatment, dilute alkali pretreatment, or solvent exposure),physical pretreatment (e.g., steam explosion or irradiation), mechanicalpretreatment (e.g., grinding or milling) and biological pretreatment(e.g., application of lignin-solubilizing microorganisms) andcombinations thereof, to increase the susceptibility of cellulose tohydrolysis.

In some embodiments, the substrate is slurried prior to pretreatment. Insome embodiments, the consistency of the slurry is between about 2% andabout 30% and more typically between about 4% and about 15%. In someembodiments, the slurry is subjected to a water and/or acid soakingoperation prior to pretreatment. In some embodiments, the slurry isdewatered using any suitable method to reduce steam and chemical usageprior to pretreatment. Examples of dewatering devices include, but arenot limited to pressurized screw presses (See e.g., WO 2010/022511,incorporated herein by reference) pressurized filters and extruders.

In some embodiments, the pretreatment is carried out to hydrolyzehemicellulose, and/or a portion thereof present in lignocellulose tomonomeric pentose and hexose sugars (e.g., xylose, arabinose, mannose,galactose, and/or any combination thereof). In some embodiments, thepretreatment is carried out so that nearly complete hydrolysis of thehemicellulose and a small amount of conversion of cellulose to glucoseoccurs. In some embodiments, an acid concentration in the aqueous slurryfrom about 0.02% (w/w) to about 2% (w/w), or any amount therebetween, istypically used for the treatment of the cellulosic substrate. Anysuitable acid finds use in these methods, including but not limited to,hydrochloric acid, nitric acid, and/or sulfuric acid. In someembodiments, the acid used during pretreatment is sulfuric acid. Steamexplosion is one method of performing acid pretreatment of biomasssubstrates (See e.g., U.S. Pat. No. 4,461,648). Another method ofpretreating the slurry involves continuous pretreatment (i.e., thecellulosic biomass is pumped though a reactor continuously). Thismethods are well-known to those skilled in the art (See e.g., U.S. Pat.No. 7,754,457).

In some embodiments, alkali is used in the pretreatment. In contrast toacid pretreatment, pretreatment with alkali may not hydrolyze thehemicellulose component of the biomass. Rather, the alkali reacts withacidic groups present on the hemicellulose to open up the surface of thesubstrate. In some embodiments, the addition of alkali alters thecrystal structure of the cellulose so that it is more amenable tohydrolysis. Examples of alkali that find use in the pretreatmentinclude, but are not limited to ammonia, ammonium hydroxide, potassiumhydroxide, and sodium hydroxide. One method of alkali pretreatment isAmmonia Freeze Explosion, Ammonia Fiber Explosion or Ammonia FiberExpansion (“AFEX” process; See e.g., U.S. Pat. Nos. 5,171,592;5,037,663; 4,600,590; 6,106,888; 4,356,196; 5,939,544; 6,176,176;5,037,663; and 5,171,592). During this process, the cellulosic substrateis contacted with ammonia or ammonium hydroxide in a pressure vessel fora sufficient time to enable the ammonia or ammonium hydroxide to alterthe crystal structure of the cellulose fibers. The pressure is thenrapidly reduced, which allows the ammonia to flash or boil and explodethe cellulose fiber structure. In some embodiments, the flashed ammoniais then recovered using methods known in the art. In some alternativemethods, dilute ammonia pretreatment is utilized. The dilute ammoniapretreatment method utilizes more dilute solutions of ammonia orammonium hydroxide than AFEX (See e.g., WO 2009/045651 and US2007/0031953). This pretreatment process may or may not produce anymonosaccharides.

An additional pretreatment process for use in the present inventionincludes chemical treatment of the cellulosic substrate with organicsolvents, in methods such as those utilizing organic liquids inpretreatment systems (See e.g., U.S. Pat. No. 4,556,430). These methodshave the advantage that the low boiling point liquids easily can berecovered and reused. Other pretreatments, such as the Organosolv™process, also use organic liquids (See e.g., U.S. Pat. No. 7,465,791).Subjecting the substrate to pressurized water may also be a suitablepretreatment method (See e.g., Weil et al., Appl. Biochem. Biotechnol.,68: 21-40 [1997]). In some embodiments, the pretreated cellulosicbiomass is processed after pretreatment by any of several steps, such asdilution with water, washing with water, buffering, filtration, orcentrifugation, or any combination of these processes, prior toenzymatic hydrolysis, as is familiar to those skilled in the art. Thepretreatment produces a pretreated feedstock composition (e.g., a“pretreated feedstock slurry”) that contains a soluble componentincluding the sugars resulting from hydrolysis of the hemicellulose,optionally acetic acid and other inhibitors, and solids includingunhydrolyzed feedstock and lignin. In some embodiments, the solublecomponents of the pretreated feedstock composition are separated fromthe solids to produce a soluble fraction. In some embodiments, thesoluble fraction, including the sugars released during pretreatment andother soluble components (e.g., inhibitors), is then sent tofermentation. However, in some embodiments in which the hemicellulose isnot effectively hydrolyzed during the pretreatment one or moreadditional steps are included (e.g., a further hydrolysis step(s) and/orenzymatic treatment step(s) and/or further alkali and/or acid treatment)to produce fermentable sugars. In some embodiments, the separation iscarried out by washing the pretreated feedstock composition with anaqueous solution to produce a wash stream and a solids stream comprisingthe unhydrolyzed, pretreated feedstock. Alternatively, the solublecomponent is separated from the solids by subjecting the pretreatedfeedstock composition to a solids-liquid separation, using any suitablemethod (e.g., centrifugation, microfiltration, plate and framefiltration, cross-flow filtration, pressure filtration, vacuumfiltration, etc.). Optionally, in some embodiments, a washing step isincorporated into the solids-liquids separation. In some embodiments,the separated solids containing cellulose, then undergo enzymatichydrolysis with cellulase enzymes in order to convert the cellulose toglucose. In some embodiments, the pretreated feedstock composition isfed into the fermentation process without separation of the solidscontained therein. In some embodiments, the unhydrolyzed solids aresubjected to enzymatic hydrolysis with cellulase enzymes to convert thecellulose to glucose after the fermentation process. In someembodiments, the pretreated cellulosic feedstock is subjected toenzymatic hydrolysis with cellulase enzymes.

As used herein, the term “chemical treatment” refers to any chemicalpretreatment that promotes the separation and/or release of cellulose,hemicellulose, and/or lignin.

As used herein, the term “physical pretreatment” refers to anypretreatment that promotes the separation and/or release of cellulose,hemicellulose, and/or lignin from cellulosic material.

As used herein, the term “mechanical pretreatment” refers to anymechanical means for treating biomass, including but not limited tovarious types of grinding or milling (e.g., dry milling, wet milling, orvibratory ball milling).

As used herein, the term “biological pretreatment” refers to anybiological pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin from cellulosic material.

As used herein, the term “recovered” refers to the harvesting,isolating, collecting, or recovering of protein from a cell and/orculture medium. In the context of saccharification, it is used inreference to the harvesting of fermentable sugars produced during thesaccharification reaction from the culture medium and/or cells. In thecontext of fermentation, it is used in reference to harvesting thefermentation product from the culture medium and/or cells. Thus, aprocess can be said to comprise “recovering” a product of a reaction(such as a soluble sugar recovered from saccharification) if the processincludes separating the product from other components of a reactionmixture subsequent to at least some of the product being generated inthe reaction.

As used herein, “increasing” the yield of a product (such as afermentable sugar) from a reaction occurs when a particular component ofinterest is present during the reaction (e.g., enzyme) causes moreproduct to be produced, compared with a reaction conducted under thesame conditions with the same substrate and other substituents, but inthe absence of the component of interest (e.g., without enzyme).

As used herein, a reaction is said to be “substantially free” of aparticular enzyme if the amount of that enzyme compared with otherenzymes that participate in catalyzing the reaction is less than about2%, about 1%, or about 0.1% (wt/wt).

As used herein, “fractionating” a liquid (e.g., a culture broth) meansapplying a separation process (e.g., salt precipitation, columnchromatography, size exclusion, and filtration) or a combination of suchprocesses to provide a solution in which a desired protein (e.g., acellulase enzyme, and/or a combination thereof) comprises a greaterpercentage of total protein in the solution than in the initial liquidproduct.

As used herein, the term “enzymatic hydrolysis,” refers to thehydrolysis of a substrate by an enzyme. In some embodiments, thehydrolysis comprises methods in which at least one enzyme is contactedwith at least one substrate to produce an end product. In someembodiments, the enzymatic hydrolysis methods comprise at least onecellulase and at least one glycosidase enzyme and/or a mixtureglycosidases that act on polysaccharides, (e.g., cellulose), to convertall or a portion thereof to fermentable sugars. “Hydrolyzing” and/or“hydrolysis” of cellulose or other polysaccharide occurs when at leastsome of the glycosidic bonds between two monosaccharides present in thesubstrate are hydrolyzed, thereby detaching from each other the twomonomers that were previously bonded.

It is intended that the enzymatic hydrolysis be carried out with anysuitable type of enzyme(s) capable of hydrolyzing at least one substrateto at least one end-product. In some embodiments, the substrate iscellulose, while in some other embodiments, it is lignocelluloses, andin still further embodiments, it is another composition (e.g., starch).In some embodiments, the end-product comprises at least one fermentablesugar. It is further intended that the enzymatic hydrolysis encompassprocesses carried out with any suitable type of cellulase enzymescapable of hydrolyzing the cellulose to glucose, regardless of theirsource. It is intended that any suitable source of enzyme will find usein the present invention, including but not limited to enzymes obtainedfrom fungi, such as Trichoderma spp., Aspergillus spp., Hypocrea spp.,Humicola spp., Neurospora spp., Orpinomyces spp., Gibberella spp.,Emericella spp., Chaetomium spp., Chrysosporium spp., Fusarium spp.,Penicillium spp., Magnaporthe spp., Phanerochaete spp., Trametes spp.,Lentinula edodes, Gleophyllum trabeiu, Ophiostoma piliferum, Corpinuscinereus, Geomyces pannorum, Cryptococcus laurentii, Aureobasidiumpullulans, Amorphotheca resinae, Leucosporidium scotti, Cunninghamellaelegans, Thermomyces lanuginosus, Myceliopthora thermophila, andSporotrichum thermophile, as well as those obtained from bacteria of thegenera Bacillus, Thermomyces, Clostridium, Streptomyces andThermobifida.

In some embodiments, the enzymatic hydrolysis is carried out at a pH andtemperature that is at or near the optimum for the cellulase enzymesbeing used. For example, in some embodiments, the enzymatic hydrolysisis carried out at about 30° C. to about 75° C., or any suitabletemperature therebetween, for example a temperature of about 30° C.,about 35° C., about 40° C., about 45° C., about 50° C., about 55° C.,about 60° C., about 65° C., about 70° C., about 75° C., or anytemperature therebetween, and a pH of about 3.5 to about 7.5, or any pHtherebetween (e.g., about 3.5, about 4.0, about 4.5, about 5.0, about5.5, about 6.0, about 6.5, about 7.0, about 7.5, or any suitable pHtherebetween). In some embodiments, the initial concentration ofcellulose, prior to the start of enzymatic hydrolysis, is preferablyabout 0.1% (w/w) to about 20% (w/w), or any suitable amount therebetween(e.g., about 0.1%, about 0.5%, about 1%, about 2%, about 4%, about 6%,about 8%, about 10%, about 12%, about 14%, about 15%, about 18%, about20%, or any suitable amount therebetween). In some embodiments, thecombined dosage of all cellulase enzymes is about 0.001 to about 100 mgprotein per gram cellulose, or any suitable amount therebetween (e.g.,about 0.001, about 0.01, about 0.1, about 1, about 5, about 10, about15, about 20, about 25, about 30, about 40, about 50, about 60, about70, about 80, about 90, about 100 mg protein per gram cellulose or anyamount therebetween). The enzymatic hydrolysis is carried out for anysuitable time period. In some embodiments, the enzymatic hydrolysis iscarried out for a time period of about 0.5 hours to about 200 hours, orany time therebetween (e.g., about 2 hours to about 100 hours, or anysuitable time therebetween). For example, in some embodiments, it iscarried out for about 0.5, about 1, about 2, about 5, about 7, about 10,about 12, about 14, about 15, about 20, about 25, about 30, about 35,about 40, about 45, about 50, about 55, about 60, about 65, about 70,about 75, about 80, about 85, about 90, about 95, about 100, about 120,about 140, about 160, about 180, about 200, or any suitable timetherebetween.

In some embodiments, the enzymatic hydrolysis is batch hydrolysis,continuous hydrolysis, and/or a combination thereof. In someembodiments, the hydrolysis is agitated, unmixed, or a combinationthereof. The enzymatic hydrolysis is typically carried out in ahydrolysis reactor. The cellulase enzyme composition is added to thepretreated lignocellulosic substrate prior to, during, or after theaddition of the substrate to the hydrolysis reactor. Indeed it is notintended that reaction conditions be limited to those provided herein,as modifications are well-within the knowledge of those skilled in theart. In some embodiments, following cellulose hydrolysis, any insolublesolids present in the resulting lignocellulosic hydrolysate, includingbut not limited to lignin, are removed using conventional solid-liquidseparation techniques prior to any further processing. In someembodiments, these solids are burned to provide energy for the entireprocess.

As used herein, the “total available cellulose” is the amount (wt %) ofcellulose that is accessible to enzymatic hydrolysis. Total availablecellulose is typically equal to, or very close to being equal to, theamount of initial cellulose present in a hydrolysis reaction.

As used herein, the “residual cellulose” is the portion (wt %) of thetotal available cellulose in the hydrolysis mixture that remainsunhydrolyzed. Residual cellulose can be measured using any suitablemethod known in the art. For example, it can be directly measured usingIR spectroscopy, or it can be measured by determining the amount ofglucose generated by concentrated acid hydrolysis of the residualsolids.

As used herein, the “total hydrolyzed cellulose” is the portion of thetotal available cellulose that is hydrolyzed in the hydrolysis mixture.For example, the total hydrolyzed cellulose can be calculated as thedifference between the “total available cellulose” and the “residualcellulose.”

As used herein, the “theoretical maximum glucose yield” is the maximumamount (wt %) of glucose that could be produced under given conditionsfrom the total available cellulose.

As used herein, “Gmax” refers to the maximum amount (wt %) of glucosethat could be produced from the total hydrolyzed cellulose. Gmax can becalculated, for example, by directly measuring the amount of residualcellulose remaining at the end of a reaction under a given reactionconditions, subtracting the amount of residual cellulose from the totalavailable cellulose to determine the total hydrolyzed cellulose, andthen calculating the amount of glucose that could be produced from thetotal hydrolyzed cellulose.

It will be appreciated by those skilled in the art that when calculatingtheoretical values such as Gmax and theoretical maximum glucose yield,the mass of two hydrogen atoms and one oxygen atom that are added to theglucose molecule in the course of the hydrolysis reaction are taken intoaccount. For example, when a polymer of “n” glucose units is hydrolyzed,(n−1) units of water are added to the glucose molecules formed in thehydrolysis, so the weight of the glucose produced is about 10% greaterthan the weight of cellulose consumed in the hydrolysis (e.g.,hydrolysis of 1 g cellulose would produce about 1.1 g glucose).

Thus, as an example, where 5 g of total available cellulose is presentat the beginning of a hydrolysis reaction, and 2 g of residual celluloseremains after the reaction, the total hydrolyzed cellulose is 3 gcellulose. A theoretical maximum glucose yield of 100% (w/w) under thereaction conditions is about 5.5 g of glucose. Gmax is calculated basedon the 3 g of cellulose that was released or converted in the reactionby hydrolysis. Thus, in this example, a Gmax of 100% (w/w) is about 3.3g of glucose. Cellulose levels, either the total available amountpresent in the substrate or the amount of unhydrolyzed or residualcellulose, can be quantified by any of a variety of methods known in theart, such as by IR spectroscopy or by measuring the amount of glucosegenerated by concentrated acid hydrolysis of the cellulose (See e.g.,U.S. Pat. Nos. 6,090,595 and 7,419,809).

As used herein, the term “undissolved solids” refers to solid materialwhich is suspended, but not dissolved, in a liquid. As is well known inthe art, the concentration of suspended or undissolved solids can bedetermined by any suitable method (e.g., by filtering a sample of theslurry using glass microfiber filter paper, washing the filter cake withwater, and drying the cake overnight at about 105° C.).

As used herein, the terms “unhydrolyzed solids,” “unconverted solids,”and the like refer to cellulose that is not digested by the cellulaseenzyme(s), as well as non-cellulosic, or other, materials that are inertto the cellulase enzyme(s), present in the feedstock.

As used herein, the term “by-product” refers to an organic molecule thatis an undesired product of a particular process (e.g.,saccharification).

In some embodiments, the present invention provides fungal organisms andmethods for the conversion of cellulose to glucose. In some embodiments,the conversion is improved by genetically modifying a fungus to reducethe amount of endogenous glucose and/or cellobiose oxidizing enzymeactivity that is secreted by the cell. Prior to this invention, it wasgenerally believed that cellobiose dehydrogenase enhances the rate ofcellulose hydrolysis by reducing the concentration of cellobiose, whichis a potent inhibitor of some cellulase components (See e.g., Mansfieldet al., Appl. Environ. Microbiol., 63: 3804-3809 [1997]; Igarishi etal., Eur. J. Biochem., 253: 101-106 [1998]). Further, cellobiosedehydrogenase has been reported as playing a critical role contributingto synergistic enhancement in degradation of cellulose by preventingproduct inhibition of hydrolysis (See e.g., Hai et al., J. Appl.Glycosci., 49:9-17 [2002]). As a result, genetic modification ofTrametes versicolor (See, Archibald, 7^(th) International Conference onBiotechnology in the Pulp and Paper Industry, Vol. B: B225-B228 [1998])and Coriolus hirsutus (See, U.S. Pat. Appln. Publn. No. 2005/0181485)has been carried out in order to produce cellulase systems with reducedcellolytic activities for pulp and paper applications. It was alsogenerally believed that cellobiose dehydrogenase was useful indelignifying lignocellulose, and thereby enhance cellulose degradation.Recently, it has been reported that cellobiose dehydrogenases canenhance the activity of cellulolytic enhancing proteins from GlycosylHydrolase Family 61 (See e.g., WO 2010/080532A1).

Contrary to general understanding in the art, the present inventionprovides fungal cells with genetic modification (such as deletion) ofglucose and/or cellobiose oxidizing enzyme-encoding genes incellulase-producing fungal cells that results in an improvement in theyield of fermentable sugars from enzyme mixtures secreted by thegenetically modified cells. Thus, reduction of glucose and/or cellobioseoxidizing enzyme activity secreted by a cellulase-producing organismresults in a mixture of cellulase enzymes that can improve yield offermentable sugars during enzymatic hydrolysis of cellulose-containingsubstrates.

The present invention provides a fungal cell that has been geneticallymodified to reduce the amount of endogenous glucose and/or cellobioseoxidizing enzyme activity that is secreted by the cell, wherein thefungal cell is an Ascomycete belonging to the subdivisionPezizomycotina, and where the fungal cell is capable of secreting acellulase-containing enzyme mixture. Also provided herein is a fungalcell that has been genetically modified to reduce the amount ofendogenous glucose and/or cellobiose oxidizing enzyme activity that issecreted by the cell and to increase the expression of at least onesaccharide hydrolyzing enzyme, wherein the fungal cell is aBasidiomycete belonging to the class Agaricomycetes, and where thefungal cell is capable of secreting a cellulase-containing enzymemixture. Also provided herein is a fungal cell that has been geneticallymodified to reduce the amount of endogenous glucose and/or cellobioseoxidizing enzyme activity that is secreted by the cell, wherein thefungal cell is a Basidiomycete, and where the fungal cell is the fungalcell is capable of secreting a cellulase-containing enzyme mixture. Insome embodiments, the endogenous glucose and/or cellobiose oxidizingenzyme is a cellobiose dehydrogenase, while in some alternativeembodiments, the endogenous glucose and/or cellobiose oxidizing enzymeis an enzyme other than cellobiose dehydrogenase.

In some embodiments, the fungal cell is capable of secreting an enzymemixture comprising at least two or more cellulase enzymes. In someembodiments, the Basidiomycete is a species of Pleurotus, Peniophora,Trametes, Athelia, Sclerotium, Termitomyces, Flammulina, Chrysosporium,Coniphora, Ganoderma, Pycnoporus, Ceriporiopsis, Phanerochaete,Gloeophyllum, Hericium, Heterobasidion, Gelatoporia, Lepiota, or Irpex.In some embodiments, the Ascomycete is a species of Myceliophthora,Thielavia, Sporotrichum, Neurospora, Sordaria, Podospora, Magnaporthe,Fusarium, Gibberella, Botryotinia, Humicola, Neosartorya, Pyrenophora,Phaeosphaeria, Sclerotinia, Chaetomium, Nectria, Verticillium, orAspergillus.

The present invention also provides a fungal cell that has beengenetically modified to reduce the amount of endogenous glucose and/orcellobiose oxidizing enzyme activity that is secreted by the cell,wherein the fungal cell is a Basidiomycete species Pleurotus,Peniophora, Athelia, Sclerotium, Termitomyces, Flammulina, Coniphora,Ganoderma, Pycnoporus, Ceriporiopsis, Phanerochaete, Gloeophyllum,Hericium, Heterobasidion, Gelatoporia, Lepiota, or Irpex.

In some embodiments, the fungal cell has been genetically modified toreduce the amount of endogenous cellobiose dehydrogenase activity thatis secreted by the cell. In some embodiments, the fungal cell has beengenetically modified to reduce the amount of endogenous glucose oxidaseactivity that is secreted by the cell. In some embodiments, the fungalcell has been genetically modified to reduce the amount of endogenouspyranose oxidase activity that is secreted by the cell. In someembodiments, the fungal cell has been genetically modified to reduce theamount of endogenous glucooligosaccharide oxidase activity that issecreted by the cell. In some embodiments, the fungal cell has beengenetically modified to reduce the amount of endogenous pyranosedehydrogenase activity that is secreted by the cell. In someembodiments, the fungal cell has been genetically modified to reduce theamount of endogenous glucose dehydrogenase activity that is secreted bythe cell.

In some embodiments, the fungal cell is a species of Myceliophthora,Thielavia, Chrysosporium, Sporotrichum, Corynascus, Acremonium,Chaetomium, Ctenomyces, Scytalidium, Talaromyces, or Thermoascus. Insome embodiments the fungal cell is a species of Myceliophthora,Thielavia, Sporotrichum, Corynascus, Acremonium, Chaetomium, orTalaromyces. In some embodiments, the fungal cell is Sporotrichumthermophile, Sporotrichum cellulophilum, Thielavia terrestris,Corynascus heterothallicus, Thielavia heterothallica, Chaetomiumglobosum, Talaromyces stipitatus, or Myceliophthora thermophila. In someembodiments, the fungal cell is an isolated fungal cell.

In some embodiments, the fungal cell has been genetically modified toreduce the amount of the endogenous glucose and/or cellobiose oxidizingenzyme that is secreted by the cell. Thus, in some embodiments, thefungal cell has been genetically modified to disrupt the secretionsignal peptide of the glucose and/or cellobiose oxidizing enzyme. Insome embodiments, the fungal cell has been genetically modified toreduce the amount of the endogenous glucose and/or cellobiose oxidizingenzyme that is expressed by the cell. For example, the fungal cell canbe genetically modified to disrupt a translation initiation sequence orto introduce a frameshift mutation in the transcript encoding theendogenous glucose and/or cellobiose oxidizing enzyme. In some otherembodiments, the fungal cell has been genetically modified to reduce thetranscription level of a gene encoding the endogenous glucose and/orcellobiose oxidizing enzyme. For example, the fungal cell can begenetically modified to disrupt the promoter of a gene encoding theendogenous glucose and/or cellobiose oxidizing enzyme. In someembodiments, the fungal cell has been genetically modified to at leastpartially delete a gene encoding the endogenous glucose and/orcellobiose oxidizing enzyme. In some other embodiments, the fungal cellhas been genetically modified to reduce the catalytic efficiency of theendogenous glucose and/or cellobiose oxidizing enzyme. In someembodiments, the fungal cell has been genetically modified to mutate oneor more residues in an active site of the glucose and/or cellobioseoxidizing enzyme. In some embodiments, the fungal cell has beengenetically modified to mutate one or more residues in a heme bindingdomain of the glucose and/or cellobiose oxidizing enzyme.

In some embodiments, the glucose and/or cellobiose oxidizing enzyme isglucose oxidase (EC 1.1.3.4). In some other embodiments, the glucoseand/or cellobiose oxidizing enzyme is cellobiose dehydrogenase (EC1.1.99.18). In some other embodiments, the glucose and/or cellobioseoxidizing enzyme is pyranose oxidase (EC1.1.3.10). In some otherembodiments, the glucose and/or cellobiose oxidizing enzyme isglucooligosaccharide oxidase (EC 1.1.99.B3). In some additionalembodiments, the glucose and/or cellobiose oxidizing enzyme is pyranosedehydrogenase (EC 1.1.99.29). In some further embodiments, the glucoseand/or cellobiose oxidizing enzyme is glucose dehydrogenase (EC1.1.99.10). In some embodiments, the glucose and/or cellobiose oxidizingenzyme comprises an amino acid sequence that is at least about 85%,about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,or at least about 99% identical to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14,and/or 16.

In some embodiments, the cell has been genetically modified to reducethe amount of glucose and/or cellobiose oxidizing enzyme activity of twoor more endogenous glucose and/or cellobiose oxidizing enzymes that aresecreted by the cell. In certain such embodiments, a first of the two ormore the glucose and/or cellobiose oxidizing enzymes comprises an aminoacid sequence that is at least about 85%, about 86%, about 87%, about88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, or at least about 99%identical to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, and/or 16, and a secondof the two or more the glucose and/or cellobiose oxidizing enzymescomprises an amino acid sequence that is at least about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, or at leastabout 99% identical to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, and/or 16.

In some embodiments, the fungal cell further comprises at least one geneencoding at least one cellulose degrading enzyme that is heterologous tothe fungal cell. For example, the fungal cell can overexpress ahomologous or heterologous gene encoding a cellulose degrading enzymesuch as beta-glucosidase. In some embodiments, the fungal celloverexpresses beta-glucosidase and has been genetically modified toreduce the amount of endogenous glucose and/or cellobiose oxidizingenzyme activity that is secreted by the cell.

The present invention also provides enzyme mixtures comprising two ormore cellulose hydrolyzing enzymes, wherein at least one of the two ormore cellulose hydrolyzing enzymes is expressed by a fungal cell asdescribed herein. For example, in some embodiments, the fungal cell is acell that has been genetically modified to reduce the amount ofendogenous glucose and/or cellobiose oxidizing enzyme activity that issecreted by the cell, wherein the fungal cell is an Ascomycete belongingto the subdivision Pezizomycotina. In some other embodiments, the fungalcell has been genetically modified to reduce the activity of anendogenous glucose and/or cellobiose oxidizing enzyme that is secretedby the cell and to increase the expression of at least one saccharidehydrolyzing enzyme, wherein the fungal cell is a Basidiomycete belongingto the class Agaricomycetes. In some embodiments, the Basidiomycete is aspecies of Pleurotus, Peniophora, Trametes, Athelia, Sclerotium,Termitomyces, Flammulina, Coniphora, Ganoderma, Pycnoporus,Ceriporiopsis, Phanerochaete, Gloeophyllum, Hericium, Heterobasidion,Gelatoporia, Lepiota, or Irpex. In some embodiments, the Ascomycete is aspecies of Myceliophthora, Thielavia, Sporotrichum, Neurospora,Sordaria, Podospora, Magnaporthe, Fusarium, Gibberella, Botryotinia,Humicola, Neosartorya, Pyrenophora, Phaeosphaeria, Sclerotinia,Chaetomium, Nectria, Verticillium, or Aspergillus. In some embodiments,the fungal cell can be a species of Myceliophthora, Thielavia,Sporotrichum, Corynascus, Acremonium, Chaetomium, Ctenomyces,Scytalidium, Talaromyces, or Thermoascus. In some embodiments the fungalcell is a species of Myceliophthora, Thielavia, Sporotrichum,Corynascus, Acremonium, or Chaetomium. In some embodiments, the fungalcell is Sporotrichum cellulophilum, Thielavia terrestris, Corynascusheterothallicus, Thielavia heterothallica, or Myceliophthorathermophila.

In some embodiments, the fungal cell is a species of Myceliophthora,Thielavia, Sporotrichum, Chrysoporium, Corynascus, Acremonium,Chaetomium, Ctenomyces, Scytalidium, Talaromyces, or Thermoascus. Insome embodiments the fungal cell is a species of Myceliophthora,Chrysosporium, Thielavia, Sporotrichum, Corynascus, Acremonium, orChaetomium. In some embodiments, the fungal cell is Sporotrichumcellulophilum, Thielavia terrestris, Corynascus heterothallicus,Thielavia heterothallica, Chaetomium globosum, Talaromyces stipitatus,or Myceliophthora thermophila. In some embodiments, the fungal cell isan isolated fungal cell.

In some additional embodiments, the enzyme mixture is a cell-freemixture. In some embodiments, a substrate of the enzyme mixturecomprises pretreated lignocellulose. In some additional embodiments, thepretreated lignocellulose comprises lignocellulose treated by atreatment method selected from acid pretreatment, ammonia pretreatment,steam explosion, organic solvent extraction, and/or any other suitablepretreatment method(s). In some embodiments, the enzyme mixture furthercomprises a cellulose degrading enzyme that is heterologous to thefungal cell. In some embodiments, at least one of the two or morecellulose hydrolyzing enzymes is expressed by an isolated fungal cell.

The present invention also provides methods for generating cellobioseand/or glucose comprising contacting a cellulosic substrate with theenzyme mixture described herein. For example, in some embodiments, themethods comprise contacting cellulose with an enzyme mixture comprisingtwo or more cellulose hydrolyzing enzymes, wherein at least one of thetwo or more cellulose hydrolyzing enzymes is expressed by a fungal cellas described herein. In some embodiments, the methods comprisecontacting cellulose with an enzyme mixture comprising two or morecellulose hydrolyzing enzymes, wherein at least one of the two or morecellulose hydrolyzing enzymes is expressed by a cell that has beengenetically modified to reduce the amount of endogenous glucose and/orcellobiose oxidizing enzyme activity that is secreted by the cell,wherein the fungal cell is a an Ascomycete belonging to the subdivisionPezizomycotina.

In some other embodiments, the fungal cell has been genetically modifiedto reduce the activity of an endogenous glucose and/or cellobioseoxidizing enzyme that is secreted by the cell and to increase theexpression of at least one saccharide hydrolyzing enzyme, wherein thefungal cell is a Basidiomycete belonging to the class Agaricomycetes. Insome embodiments, the Basidiomycete is a species of Pleurotus,Peniophora, Trametes, Athelia, Sclerotium, Termitomyces, Flammulina,Coniphora, Ganoderma, Pycnoporus, Ceriporiopsis, Phanerochaete,Gloeophyllum, Hericium, Heterobasidion, Gelatoporia, Lepiota, or Irpex.In some embodiments, the Ascomycete is a species of Myceliophthora,Thielavia, Sporotrichum, Neurospora, Sordaria, Podospora, Magnaporthe,Fusarium, Gibberella, Botryotinia, Humicola, Neosartorya, Pyrenophora,Phaeosphaeria, Sclerotinia, Chaetomium, Nectria, Verticillium, orAspergillus. In some embodiments, the fungal cell is a species ofMyceliophthora, Thielavia, Sporotrichum, Corynascus, Acremonium,Chaetomium, Ctenomyces, Scytalidium, Talaromyces, or Thermoascus. Insome embodiments the fungal cell is a species of Myceliophthora,Thielavia, Sporotrichum, Corynascus, Acremonium, or Chaetomium. In someembodiments, the fungal cell is Sporotrichum cellulophilum, Thielaviaterrestris, Corynascus heterothallicus, Thielavia heterothallica, orMyceliophthora thermophila.

In some embodiments, the fungal cell is a species of Myceliophthora,Thielavia, Sporotrichum, Corynascus, Acremonium, Chaetomium, Ctenomyces,Scytalidium, Talaromyces, or Thermoascus. In some embodiments the fungalcell is a species of Myceliophthora, Thielavia, Sporotrichum,Corynascus, Acremonium, or Chaetomium. In some embodiments, the fungalcell is Sporotrichum cellulophilum, Thielavia terrestris, Corynascusheterothallicus, Thielavia heterothallica, Chaetomium globosum,Talaromyces stipitatus, or Myceliophthora thermophila. In someembodiments, the fungal cell is an isolated fungal cell.

The present invention provides methods for generating cellobiose and/orglucose comprising contacting a cellulose substrate with an enzymemixture comprising two or more cellulose hydrolyzing enzymes to generateglucose and/or cellobiose, wherein at least one of the cellulosehydrolyzing enzymes is endogenous to a fungus that is an Ascomycetebelonging to the subdivision Pezizomycotina, and wherein the enzymemixture is characterized in that, when the enzyme mixture is contactedwith cellobiose and/or glucose, no more than about 1%, about 2%, about3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%,about 17%, about 18%, about 19%, or about 20% (wt %) of the cellobioseand/or glucose is oxidized after 10 hours.

The present invention also provides methods for generating cellobioseand/or glucose comprising contacting a cellulose substrate with anenzyme mixture comprising two or more cellulose hydrolyzing enzymes togenerate glucose and/or cellobiose, wherein at least one of thecellulose hydrolyzing enzymes is endogenous to a fungus that is aBasidiomycete belonging to the class Agaricomycetes, and wherein theenzyme mixture is characterized in that, when the enzyme mixture iscontacted with cellobiose and/or glucose, no more than about 1%, about2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%,about 16%, about 17%, about 18%, about 19%, or about 20% (wt %) of thecellobiose and/or glucose is oxidized after 10 hours.

The present invention further provides methods for generating cellobioseand/or glucose comprising contacting a cellulose substrate with anenzyme mixture comprising two or more cellulose hydrolyzing enzymes togenerate glucose and/or cellobiose, wherein at least one of thecellulose hydrolyzing enzymes is endogenous to a fungus of a species ofMyceliophthora, Thielavia, Sporotrichum, Corynascus, Acremonium,Chaetomium or Ctenomyces, Scytalidium or Thermoascus, and wherein theenzyme mixture is characterized in that, when the enzyme mixture iscontacted with cellobiose and/or glucose, no more than about 1%, about2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%,about 16%, about 17%, about 18%, about 19%, or about 20% (wt %) of thecellobiose and/or glucose is oxidized after 10 hours.

In some embodiments of the methods provided herein, when the enzymemixture is contacted with a cellulose substrate, no more than about 1%,about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%,about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about15%, about 16%, about 17%, about 18%, about 19%, or 20% (wt %) of thecellobiose and/or glucose resulting from the hydrolysis of the cellulosesubstrate is oxidized. For example, when the enzyme mixture is contactedwith a cellulose substrate, no more than about 1%, about 2%, about 3%,about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%,about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about17%, about 18%, about 19% or about 20% (wt %) of the cellobiose and/orglucose resulting from the hydrolysis of the cellulose substrate isoxidized to form cellobionolactone, cellobionic acid, gluconolactone,gluconate or gluconic acid after a period of time during whichhydrolysis occurs. For example, no more than about 1%, about 2%, about3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%,about 17%, about 18%, about 19%, or about 20% (wt %) of cellobioseand/or glucose is oxidized after about 1, about 5, about 10, about 20,about 30, about 40, about 50 or about 60 minutes, or after about 1.5,about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9,about 10, about 12, about 14, about 16, about 18, about 20, about 25,about 30, about 35, about 40, about 45, about 50, about 55, about 60,about 65, about 70, about 75, about 80, about 85, about 90, about 95,about 100, about 105, about 110, about 115, about 120, about 125, about130, about 135, about 140, about 145, about 150, about 155, about 160,about 165, about 170, about 175, about 180, about 185, about 190, about195, about 200, about 205, about 210, about 215, about 220, about 225,about 230, about 235, about 240, about 245, about 250, about 255, about260, about 265, about 270, about 275, about 280, about 285, about 290,about 395, about 300 hours, or longer.

The present invention provides methods for generating cellobiose and/orglucose comprising contacting a cellulose substrate with an enzymemixture comprising two or more cellulose hydrolyzing enzymes to generateglucose and/or cellobiose, wherein at least one of the cellulosehydrolyzing enzymes is endogenous to a fungus that is an Ascomycetebelonging to the subdivision Pezizomycotina, and wherein, of thecellulose hydrolyzed by the enzyme mixture, at least about 80%, about81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 98%, about 99% or100% (wt %) is present in the form of cellobiose and/or glucose.

The present invention also provides methods for generating cellobioseand/or glucose comprising contacting a cellulose substrate with anenzyme mixture comprising two or more cellulose hydrolyzing enzymes togenerate glucose and/or cellobiose, wherein at least one of thecellulose hydrolyzing enzymes is endogenous to a fungus that is aBasidiomycete belonging to the class Agaricomycetes, and wherein, of thecellulose hydrolyzed by the enzyme mixture, at least about 80%, about81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 98%, about 99%,or about 100% (wt %) is present in the form of cellobiose and/orglucose.

In some embodiments, no more than about 1%, about 2%, about 3%, about4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%,about 18%, about 19%, or about 20% (wt %) of the cellulose hydrolyzed bythe enzyme mixture is present in the form of gluconolactone or gluconicacid. In some embodiments, no more than about 1%, about 2%, about 3%,about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%,about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about17%, about 18%, about 19%, or about 20% (wt %) of the cellulosehydrolyzed by the enzyme mixture is present in the form ofgluconolactone, gluconic acid, cellobionolactone or cellobionic acid.

Further provided herein are methods for generating cellobiose and/orglucose comprising contacting a cellulose substrate with an enzymemixture comprising two or more cellulose hydrolyzing enzymes to generateglucose and/or cellobiose, wherein at least one of the cellulosehydrolyzing enzymes is endogenous to a fungus of a species of Pleurotus,Peniophora, Trametes, Athelia, Sclerotium, Termitomyces, Flammulina,Coniphora, Ganoderma, Pycnoporus, Ceriporiopsis, Phanerochaete,Gloeophyllum, Hericium, Heterobasidion, Gelatoporia, Lepiota, or Irpex,and wherein, of the cellulose hydrolyzed by the enzyme mixture, at leastabout 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about98%, about 99%, or about 100% (wt %) is present in the form ofcellobiose and/or glucose.

Further provided herein are methods for generating cellobiose and/orglucose comprising contacting a cellulose substrate with an enzymemixture comprising two or more cellulose hydrolyzing enzymes to generateglucose and/or cellobiose, wherein at least one of the cellulosehydrolyzing enzymes is endogenous to a fungus of a species ofMyceliophthora, Thielavia, Sporotrichum, Neurospora, Sordaria,Podospora, Magnaporthe, Fusarium, Gibberella, Botryotinia, Humicola,Neosartorya, Pyrenophora, Phaeosphaeria, Sclerotinia, Chaetomium,Nectria, Verticillium, or Aspergillus, and wherein, of the cellulosehydrolyzed by the enzyme mixture, at least about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 98%, about 99%, or about100% (wt %) is present in the form of cellobiose and/or glucose.

In some embodiments, no more than about 1%, about 2%, about 3%, about4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%,about 18%, about 19%, or about 20% (wt %) of the cellulose hydrolyzed bythe enzyme mixture is present in the form of gluconolactone or gluconicacid. In some embodiments, no more than about 1%, about 2%, about 3%,about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%,about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about17%, about 18%, about 19%, or about 20% (wt %) of the cellulosehydrolyzed by the enzyme mixture is present in the form ofgluconolactone, gluconic acid, cellobionolactone or cellobionic acid.

In some embodiments, the methods result in an increased yield of glucoseand/or cellobiose from the hydrolyzed cellulose and a decreasedoxidation of the glucose and/or cellobiose to oxidized sugar products,such as gluconolactone, gluconate, gluconic acid, cellobionolactone,and/or cellobionic acid from the hydrolyzed cellulose. In someembodiments, the methods result in an increased yield of glucose and/orcellobiose from the hydrolyzed cellulose and a decreased oxidation ofthe glucose and/or cellobiose to oxidized sugar products, such asgluconolactone, gluconate, gluconic acid, cellobionolactone, and/orcellobionic acid from the hydrolyzed cellulose, relative to an enzymemixture with an unmodified amount of glucose and/or cellobiose oxidizingenzyme activity, or relative to a parental enzyme mixture.

In some embodiments, the present invention provides methods forproducing cellobiose and/or glucose from cellulose comprising treating acellulose substrate with an enzyme mixture to generate glucose and/orcellobiose, wherein the enzyme mixture is modified relative to asecreted enzyme mixture from a wild type or reference (e.g., parental)fungal cell to be at least partially deficient in glucose and/orcellobiose oxidizing enzyme activity.

In some aspects of the above embodiments, the enzyme mixture is acell-free mixture. In some other aspects, the cellulose substratecomprises pretreated lignocellulose. In some embodiments, the pretreatedlignocellulose comprises lignocellulose treated by a treatment methodselected from acid pretreatment, ammonia pretreatment, steam explosion,and organic solvent extraction.

In some aspects of the above embodiments, the methods further comprisefermentation of the glucose to an end product such as a fuel alcohol ora precursor industrial chemical. In some aspects, the fuel alcohol isethanol or butanol. Accordingly, in some embodiments, increased glucoseyield can result in lower fuel production costs. In some aspects, themethods comprise contacting cellulose with an enzyme mixture thatfurther comprises a cellulose degrading enzyme that is heterologous tothe fungal cell.

In some embodiments, the enzyme mixture is produced by a fungal cell hasthat been genetically modified to reduce the amount of one or moreendogenous glucose and/or cellobiose oxidizing enzymes that is secretedby the cell.

In some embodiments, the enzyme mixture is subjected to a purificationprocess to selectively remove one or more glucose and/or cellobioseoxidizing enzymes from the enzyme mixture. In some such aspects, thepurification process comprises selective precipitation to separate theglucose and/or cellobiose oxidizing enzymes from other enzymes presentin the enzyme mixture.

In some embodiments, the enzyme mixture comprises an inhibitor of one ormore glucose and/or cellobiose oxidizing enzymes. In some embodiments,the inhibitor includes a broad-spectrum oxidase inhibitor selected fromsodium azide, potassium cyanide and a number of metal anions such asAg⁺, Hg²⁺, Zn²⁺. In some additional embodiments, the inhibitor includesa specific inhibitor of cellobiose dehydrogenase (EC 1.1.99.18) such asgentiobiose, lactobiono-1,5-lactone, celliobono-1,5-lactone,tri-N-acetylchitortriose, methyl-beta-D-cellobioside, 2,2-bipyridine andcytochrome C.

In some embodiments, the enzyme mixture comprises at least onebeta-glucosidase. In some additional embodiments, the enzyme mixturecomprises at least one cellulase enzyme selected from endoglucanases(EGs), β-glucosidases (BGLs), Type 1 cellobiohydrolases (CBH1s), Type 2cellobiohydrolases (CBH2s), and/or glycoside hydrolase 61s (GH61s),and/or variants of said cellulase enzyme.

The present invention also provides enzyme mixtures comprising two ormore cellulose hydrolyzing enzymes, at least one of the cellulosehydrolyzing enzymes being endogenous to a fungal cell, wherein thefungal cell is an Ascomycete belonging to the subdivision Pezizomycotinaand wherein the enzyme mixtures are characterized in that, when theenzyme mixtures are contacted with cellobiose and/or glucose, no morethan about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%,about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, orabout 20% (wt %) of the cellobiose and/or glucose is oxidized after 10hours. In some aspects of the above embodiments, the fungal cell is aspecies of Myceliophthora, Thielavia, Sporotrichum, Corynascus,Acremonium, Chaetomium, Ctenomyces, Scytalidium, Talaromyces orThermoascus.

The present invention also provides enzyme mixtures comprising two ormore cellulose hydrolyzing enzymes, at least one of the cellulosehydrolyzing enzymes being endogenous to a fungal cell, wherein thefungal cell is a Basidiomycete belonging to the class Agaricomycetes andwherein the enzyme mixtures are characterized in that, when the enzymemixtures are contacted with cellobiose and/or glucose, no more thanabout 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%(wt %) of the cellobiose and/or glucose is oxidized after 10 hours.

The present invention further provides enzyme mixtures comprising two ormore cellulose hydrolyzing enzymes, at least one of the cellulosehydrolyzing enzymes being endogenous to a fungal cell, wherein thefungal cell is Pleurotus, Peniophora, Trametes, Athelia, Sclerotium,Termitomyces, Flammulina, Coniphora, Ganoderma, Pycnoporus,Ceriporiopsis, Phanerochaete, Gloeophyllum, Hericium, Heterobasidion,Gelatoporia, Lepiota, or Irpex, and wherein the enzyme mixtures arecharacterized in that, when the enzyme mixtures are contacted withcellobiose and/or glucose, no more than about 1%, about 2%, about 3%,about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%,about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about17%, about 18%, about 19%, or about 20% (wt %) of the cellobiose and/orglucose is oxidized after 10 hours.

The present invention also provides enzyme mixtures comprising two ormore cellulose hydrolyzing enzymes, at least one of the cellulosehydrolyzing enzymes being endogenous to a fungal cell, wherein thefungal cell is Myceliophthora, Thielavia, Sporotrichum, Neurospora,Sordaria, Podospora, Magnaporthe, Fusarium, Gibberella, Botryotinia,Humicola, Neosartorya, Pyrenophora, Phaeosphaeria, Sclerotinia,Chaetomium, Nectria, Verticillium, or Aspergillus, and wherein theenzyme mixtures are characterized in that, when the enzyme mixtures arecontacted with cellobiose and/or glucose, no more than about 1%, about2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%,about 16%, about 17%, about 18%, about 19%, or about 20% (wt %) of thecellobiose and/or glucose is oxidized after 10 hours.

In some aspects of the above embodiments, the fungal cell has beengenetically modified to reduce the amount of one or more endogenousglucose and/or cellobiose oxidizing enzymes that is secreted by thecell. In some embodiments, the enzyme mixtures are cell-free mixtures.In some embodiments, the enzyme mixtures contain a beta-glucosidase. Insome additional embodiments, the enzyme mixtures comprise at least onecellulase enzyme selected from endoglucanases (EGs), β-glucosidases(BGLs), Type 1 cellobiohydrolases (CBH1s), Type 2 cellobiohydrolases(CBH2s), and/or glycoside hydrolase 61s (GH61s), and/or variants of saidcellulase enzyme.

In some embodiments, the enzyme mixtures are subjected to a purificationprocess to selectively remove one or more glucose and/or cellobioseoxidizing enzymes from the enzyme mixture. In some embodiments, thepurification process comprises selective precipitation to separate theglucose and/or cellobiose oxidizing enzymes from other enzymes presentin the enzyme mixture. In some embodiments, the enzyme mixtures compriseat least one inhibitor of one or more glucose and/or cellobioseoxidizing enzymes.

The present invention also provides compositions comprising the fungalcell of any of the above embodiments, and/or comprising the enzymemixture derived from the fungal cell of any of the above embodiments.

In some embodiments, the present invention provides methods for theproduction of fungal cells. In some further embodiments, the presentinvention provides methods for the production of at least one enzymefrom fungal cells. In some embodiments, these methods comprisefermentation methods, including but not limited to, batch process,continuous process, fed-batch and/or a combination of methods. In someembodiments, the methods are conducted in a reaction volume of at leastabout 0.01 mL, about 0.1 mL, about 1 mL, about 10 mL, about 100 mL,about 1000 mL, or at least about 10 L, about 50 L, about 100 L, about200 L, about 300 L, about 400 L, about 500 L, about 600 L, about 700 L,about 800 L, about 900 L, about 1000 L, about 10,000 L, about 50,000 L,about 100,000 L, about 250,000 L, about 500,000 L, or greater than about1,000,000 L.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides genetically modified cellulase-producingfungal cells that have reduced secreted activity of an endogenousglucose and/or cellobiose oxidizing enzyme, and which are therefore ableto secrete enzyme mixtures that improve the yield of fermentable sugarsfrom cellulose. Previous reports have indicated that the oxidation ofcellobiose by cellobiose dehydrogenase enhances the rate of cellulosehydrolysis by cellulases. In contrast to the traditional thinking in theart, the present invention provides fungal cells with genomicdeletion(s) or other genetic modification(s) to reduce glucose and/orcellobiose oxidizing enzyme activity that results in improved yield offermentable sugars from cellulose. Advantageously, the geneticallymodified cellulase-producing fungal cells provided herein secrete enzymemixtures that result in vastly improved yields of fermentable sugarssuch as glucose from cellulose.

Among the cellulase-producing filamentous fungi, there are those thatalso produce a variety of enzymes involved in lignin degradation. Forexample, organisms of such genera as Myceliophthora, Chrysosporium,Sporotrichum, Thielavia, Phanerochaete and Trametes produce and secretea mixture of cellulases, hemicellulases and lignin degrading enzymes.These types of organisms are commonly called “white rot fungi” by virtueof their ability to digest lignin and to distinguish them from the“brown rot” fungi (such as Trichoderma) which typically cannot digestlignin. The genera Myceliophthora, Chrysosporium, Sporotrichum, andThielavia are closely related and in some cases different genus/speciesidentifiers have been used interchangeably for strains of the samespecies (e.g., M. thermophila and S. thermophile). Continuingdevelopments in the methods to establish the taxonomy of filamentousfungi has led to reclassification of some strains from one genus toanother or has identified an “anamorph-teleomorph” relationship betweenstrains of two genera (e.g., M. thermophila and T. heterothallica).

The present invention provides cells, enzyme mixtures and methods inwhich the activity of glucose and/or cellobiose oxidizing enzyme(s) isreduced so as to improve the yield of fermentable sugars from anenzymatic cellulose hydrolysis process. In view of this, as describedherein, the present invention provides for removal or inactivation ofglucose and/or cellobiose oxidizing enzyme from a mixture of cellulaseenzymes to improve the yield of fermentable sugars from cellulose orbiomass.

Genetically Modified Fungal Cells

The genetically modified fungal cells provided herein permit a reductionin the amount of endogenous glucose and/or cellobiose oxidizing enzymeactivity that is secreted by the cell.

In some embodiments of the genetically modified fungal cells providedherein, glucose and/or cellobiose oxidizing enzyme activity that issecreted by the cell is reduced by at least about 5%, about 10%, about15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 99%, or more, relativeto the level of glucose and/or cellobiose oxidizing enzyme activitysecreted by the unmodified parental fungal cell grown or cultured underessentially the same culture conditions.

In some embodiments, the genetic modification results in at least about5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, orabout a 99% reduction in the total glucose and/or cellobiose oxidizingenzyme activity secreted by the fungal cell.

It will be readily appreciated that any genetic modification known inthe art can be employed to reduce the secreted activity of theendogenous glucose and/or cellobiose oxidizing enzyme. For example, asdescribed below, modifications contemplated herein include modificationsthat reduce the amount of glucose and/or cellobiose oxidizing enzymesecreted by the cell. Further contemplated are modifications that reducethe amount of glucose and/or cellobiose oxidizing enzyme that isexpressed by the cell. Additional embodiments include modifications thatreduce the transcription level of glucose and/or cellobiose oxidizingenzyme. Still further embodiments include the complete or partialdeletion of a gene encoding glucose and/or cellobiose oxidizing enzyme.Other embodiments include modifications that reduce the catalyticefficiency of glucose and/or cellobiose oxidizing enzyme.

Secreted Enzyme(s). Accordingly, in some embodiments, the fungal cellhas been genetically modified to reduce the amount of the endogenousglucose and/or cellobiose oxidizing enzyme that is secreted by the cell.The glucose and/or cellobiose oxidizing enzyme that is secreted by acell is a glucose and/or cellobiose oxidizing enzyme produced by thecell in a manner such that the glucose and/or cellobiose oxidizingenzyme is exported across a cell membrane and then subsequently releasedinto the extracellular milieu, such as into culture media. Thus, areduction in the amount of secreted glucose and/or cellobiose oxidizingenzyme can be a complete or partial reduction of the glucose and/orcellobiose oxidizing enzyme secreted to the extracellular milieu.Reduction in the amount of secreted glucose and/or cellobiose oxidizingenzyme can be accomplished by reducing the amount of glucose and/orcellobiose oxidizing enzyme produced by the cell and/or by reducing theability of the cell to secrete the glucose and/or cellobiose oxidizingenzyme that is produced by the cell. Methods for reducing the ability ofthe cell to secrete a polypeptide can be performed according to any of avariety of methods known in the art (See e.g., Fass and Engels, J. Biol.Chem., 271, 15244-15252 [1996]). For example, the gene encoding asecreted polypeptide can be modified to delete or inactivate a secretionsignal peptide. Thus, in some embodiments, the fungal cell has beengenetically modified to disrupt the N-terminal secretion signal peptideof the glucose and/or cellobiose oxidizing enzyme. The amount of glucoseand/or cellobiose oxidizing enzyme that is secreted by the cell can bereduced by at least about 5%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or more, relative to the secretion ofglucose and/or cellobiose oxidizing enzyme in an unmodified organismgrown or cultured under essentially the same culture conditions.

Furthermore, the total amount of glucose and/or cellobiose oxidizingenzyme activity can be reduced by at least about 5%, about 10%, about15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 99% or more, relativeto the total amount of glucose and/or cellobiose oxidizing enzymesecreted in an unmodified organism grown or cultured under essentiallythe same culture conditions.

Decreased secretion of a glucose and/or cellobiose oxidizing enzyme canbe determined by any of a variety of methods known in the art fordetection of protein or enzyme levels. For example, the levels ofglucose and/or cellobiose oxidizing enzyme in the supernatant of afungal culture can be detected using Western blotting techniques orother protein detection techniques that use an antibody specific to theglucose and/or cellobiose oxidizing enzyme. Similarly, secreted glucoseand/or cellobiose oxidizing enzyme activity in the supernatant of afungal culture can be measured using assays for glucose and/orcellobiose oxidizing enzyme activity as described in greater detailherein.

Expression Level. In some embodiments, the fungal cell has beengenetically modified to reduce the amount of the endogenous glucoseand/or cellobiose oxidizing enzyme that is expressed by the cell. Insome embodiments, the reduction in the expression is accomplished byreducing the amount of mRNA that is transcribed from a gene encoding theglucose and/or cellobiose oxidizing enzyme. In some other embodiments,the reduction in the expression is accomplished by reducing the amountof protein that is translated from a mRNA encoding the glucose and/orcellobiose oxidizing enzyme.

The amount of glucose and/or cellobiose oxidizing enzyme that isexpressed by the cell can be reduced by at least about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more,relative to the expression of glucose and/or cellobiose oxidizing enzymein an unmodified fungal cell. In some embodiments, the reduction in theexpression is accomplished by reducing the amount of mRNA that istranscribed from a gene encoding cellobiose dehydrogenase or glucoseoxidase in an unmodified organism grown or cultured under essentiallythe same culture conditions.

Furthermore, in some embodiments a reduction in the expression level ofa glucose and/or cellobiose oxidizing enzyme will result in at leastabout 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,or about a 99% reduction in the total expression level of glucose and/orcellobiose oxidizing enzyme activity by the fungal cell relative to anunmodified fungal cell grown or cultured under essentially the sameculture conditions.

Decreased expression of a glucose and/or cellobiose oxidizing enzyme canbe determined by any of a variety of methods known in the art fordetection of protein or enzyme levels. For example, the levels ofglucose and/or cellobiose oxidizing enzyme in the supernatant of afungal culture can be detected using Western blotting techniques orother protein detection techniques that use an antibody specific to theglucose and/or cellobiose oxidizing enzyme.

Methods for reducing expression of a polypeptide are well known and canbe performed using any of a variety of methods known in the art. Forexample, the gene encoding a secreted polypeptide can be modified todisrupt a translation initiation sequence such as a Shine-Delgarnosequence or a Kozak consensus sequence. Furthermore, the gene encoding asecreted polypeptide can be modified to introduce a frameshift mutationin the transcript encoding the endogenous glucose and/or cellobioseoxidizing enzyme. It will also be recognized that usage of uncommoncodons can result in reduced expression of a polypeptide. It will beappreciated that in some embodiments, the gene encoding the glucoseand/or cellobiose oxidizing enzyme can have a nonsense mutation thatresults in the translation of a truncated protein.

Other methods of reducing the amount of expressed polypeptide includepost-transcriptional RNA silencing methodologies such as antisense RNAand RNA interference. Antisense techniques are well-established, andinclude using a nucleotide sequence complementary to the nucleic acidsequence of the gene. More specifically, in some embodiments, expressionof the gene by a fungal cell is reduced or eliminated by introducing anucleotide sequence complementary to the nucleic acid sequence, which istranscribed in the cell and is capable of hybridizing to the mRNAproduced in the cell. Under conditions allowing the complementaryanti-sense nucleotide sequence to hybridize to the mRNA, the amount ofprotein translated is thus reduced or eliminated (See e.g., Ngiam etal., Appl Environ. Microbiol., 66:775-82 [2000]; and Zrenner et al.,Planta 190:247-52 [1993]).

In some further embodiments, modification, downregulation and/orinactivation of the gene is achieved via any suitable RNA interference(RNAi) technique (See e.g., Kadotani et al. Mol. Plant MicrobeInteract., 16:769-76 [2003]). RNA interference methodologies includedouble stranded RNA (dsRNA), short hairpin RNAs (shRNAs) and smallinterfering RNAs (siRNAs). Potent silencing using dsRNA may be obtained(See e.g., Fire et al., Nature 391:806-11 [1998]). Silencing usingshRNAs is also well-established (See e.g., Paddison et al., Genes Dev.16:948-958 [2002]). Silencing using siRNA techniques are also known (Seee.g., Miyagishi et al., Nat. Biotechnol., 20:497-500 [2002].

Transcription Level. In some embodiments, the fungal cell has beengenetically modified to reduce the transcription level of a geneencoding the endogenous glucose and/or cellobiose oxidizing enzyme. Thetranscription level can be reduced by at least about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more,relative to the transcription level of a glucose and/or cellobioseoxidizing enzyme in an unmodified organism grown or cultured underessentially the same culture conditions.

Furthermore, a reduction in the transcription level of a glucose and/orcellobiose oxidizing enzyme will result in at least about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, or about a 99%reduction in the total glucose and/or cellobiose oxidizing enzymeactivity secreted by the fungal cell relative to an unmodified organismgrown or cultured under essentially the same culture conditions.

Decreased transcription can be determined by any of a variety of methodsknown in the art for detection of transcription levels. For example, thelevels of transcription of a particular mRNA in a fungal cell can bedetected using quantitative RT-PCR techniques or other RNA detectiontechniques that specifically detect a particular mRNA.

Methods for reducing transcription level of a gene can be performedaccording to any method known in the art, and include partial orcomplete deletion of the gene, and disruption or replacement of thepromoter of the gene such that transcription of the gene is greatlyreduced or even inhibited. For example, the promoter of the gene can bereplaced with a weak promoter (See e.g., U.S. Pat. No. 6,933,133). Thus,where the weak promoter is operably linked with the coding sequence ofan endogenous polypeptide, transcription of that gene will be greatlyreduced or even inhibited.

Gene Deletion. In some embodiments, the fungal cell has been geneticallymodified to at least partially delete a gene encoding the endogenousglucose and/or cellobiose oxidizing enzyme. In some embodiments, thisdeletion reduces or eliminates the total amount of endogenous glucoseand/or cellobiose oxidizing enzyme activity secreted by the fungal cell.

A deletion in a gene encoding a glucose and/or cellobiose oxidizingenzyme in accordance with the embodiments provided herein can be adeletion of one or more nucleotides in the gene encoding the glucoseand/or cellobiose oxidizing enzyme, and is often a deletion of at leastabout 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, or about 100% of the gene encoding the glucose and/orcellobiose oxidizing enzyme, where the amount of glucose and/orcellobiose oxidizing enzyme activity secreted by the cell is reduced.

Thus, for example, in some embodiments, the deletion results in at leastabout 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,or about a 99% reduction in the activity of the endogenous glucoseand/or cellobiose oxidizing enzyme secreted by the fungal cell, relativeto the activity of the glucose and/or cellobiose oxidizing enzymesecreted by an unmodified organism grown or cultured under essentiallythe same culture conditions.

Furthermore, in some embodiments, the deletion results in at least about5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, orabout a 99% reduction in the total glucose and/or cellobiose oxidizingenzyme activity secreted by the fungal cell relative to an unmodifiedfungal cell grown or cultured under essentially the same cultureconditions.

Deletion of a glucose and/or cellobiose oxidizing enzyme gene can bedetected and confirmed by any of a variety of methods known in the artfor detection of gene deletions. For example, as exemplified in theExample section below, gene deletion can be confirmed using PCRamplification of the modified genomic region. It will be appreciatedthat additional techniques for confirming deletion can be used and arewell known, including Southern blot techniques, DNA sequencing of themodified genomic region, and screening for positive or negative markersincorporated during recombination events.

Methods for complete and/or partial deletion of a gene are well-knownand the genetically modified fungal cell described herein can begenerated using any of a variety of deletion methods known in the artthat permits a reduction in the amount of endogenous glucose and/orcellobiose oxidizing enzyme activity that is secreted by the cell. Suchmethods may advantageously include standard gene disruption usinghomologous flanking markers (See e.g., Rothstein, Meth. Enzymol.,101:202-211 [1983]). Another technique for gene deletion includesPCR-based methods for standard deletion (See e.g., Davidson et al.,Microbiol., 148:2607-2615 [2002], which describes a PCR-based strategyto generate integrative targeting alleles with large regions ofhomology).

Further gene deletion techniques include “positive-negative” cassettes;cre/lox based deletion, biolistic transformation to increase homologousrecombination, and agrobacterium-mediated gene disruption. The“positive-negative” method employs cassettes which consist of one markergene for positive screening and another marker gene for negativescreening (See e.g., Chang et al., Proc. Natl. Acad. Sci. USA84:4959-4963 [1987]). Cre/lox based methodologies employ elimination ofmarker genes using expression of Cre recombinase (See e.g., Florea etal., Fung. Genet. Biol., 46:721-730 [2009]).

Methods to introduce DNA or RNA into fungal cells are known to those ofskill in the art and include PEG-mediated transformation of protoplasts,electroporation, biolistic transformation, and Agrobacterium-mediatedtransformation. Biolistic transformation employs a unique process inwhich DNA or RNA is introduced into cells on micron-sized particles,thus increasing delivery of a deletion construct to the fungal cell (Seee.g., Davidson et al., Fung. Genet. Biol., 29:38-48 [2000]. Similarly,Agrobacterium-mediated transformation in conjunction with linear orsplit-marker deletion cassettes can facilitate delivery of deletionconstructs to the target cell (See e.g., Wang et al., Curr. Genet.,56:297-307 [2010]).

Additional methods for complete or partial gene deletion include, butare not limited to, disruption of the gene. Such gene disruptiontechniques are known to those of skill in the art, and include the useof, for example, insertional mutagenesis, the use of transposons andmarked integration. However, it will be appreciated that any techniquethat provides for disruption of the coding sequence or any otherfunctional aspect of a gene can be utilized to generate the geneticallymodified fungal cells provided herein. Methods of insertionalmutagenesis can be performed according to any such method known in theart (See e.g., Combier et al., FEMS Microbiol. Lett., 220:141-8 [2003]).For example, Agrobacterium-mediated insertional mutagenesis can be usedto insert a sequence that disrupts the function of the encoded gene,such as disruption of the coding sequence or any other functional aspectof the gene.

Transposon mutagenesis methodologies are another manner for disruptionof a gene. Transposon mutagenesis is well known in the art, and can beperformed using in vivo techniques (See e.g., Firon et al., Eukaryot.Cell 2:247-55 [2003]); or by the use of in vitro techniques (See e.g.,Adachi et al., Curr Genet., 42:123-7 [2002]). The content of each ofthese references is incorporated by reference in its entirety. Thus,targeted gene disruption using transposon mutagenesis can be used toinsert a sequence that disrupts the function of the encoded gene, suchas disruption of the coding sequence or any other functional aspect ofthe gene.

Restriction enzyme-mediated integration (REMI) is another methodologyfor gene disruption, and is well known in the art (See e.g., Thon etal., Mol. Plant. Microbe Interact., 13:1356-65 [2000], which isincorporated by reference herein in its entirety). REMI generatesinsertions into genomic restriction sites in an apparently randommanner, some of which cause mutations. Thus, insertional mutants thatdemonstrate a disruption in the gene encoding the endogenous glucoseand/or cellobiose oxidizing enzyme can be selected and utilized asprovided herein.

Catalytic Disruption. In some other embodiments, the fungal cell isgenetically modified to reduce the catalytic efficiency of theendogenous glucose and/or cellobiose oxidizing enzyme. In someembodiments, a genetic modification that reduces catalytic efficiencycan result in, for example, a translated protein product that has areduction in enzymatic activity.

In some embodiments, a reduction in catalytic efficiency is a reductionof glucose and/or cellobiose oxidizing enzyme activity of about 5%,about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more, relative to unmodified glucose and/or cellobiose oxidizingenzyme, as measured using standard techniques.

In some further embodiments, the genetic modification results in areduction of glucose and/or cellobiose oxidizing enzyme activity of atleast about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, or about a 99% reduction in the total glucose and/or cellobioseoxidizing enzyme activity secreted by the fungal cell.

Methods for reducing catalytic efficiency of dehydrogenases and oxidasesare well known, and as such, any of a variety of suitable methods knownin the art for reducing catalytic efficiency can be utilized in thegenetic modification of the fungal cells provided herein. Thus, forexample, the fungal cell can be genetically modified to inactivate oneor more residues in an active site of the glucose and/or cellobioseoxidizing enzyme (See e.g., Frederik et al., Biochem., 42:4049-4056[2003]). For example, one or more residues can be modified to decreasesubstrate binding, and/or one or more residues can be modified todecrease the catalytic activity of the glucose and/or cellobioseoxidizing enzyme. Accordingly, one or more residues in the electronacceptor (e.g., flavin) binding domain, saccharide binding domain orother substrate binding domain of glucose and/or cellobiose oxidizingenzyme can be performed to reduce or inactivate the catalytic efficiencyof the glucose and/or cellobiose oxidizing enzyme. Similarly, it will beapparent that mutation of residues outside an active site can result inallosteric change in the shape or activity of the glucose and/orcellobiose oxidizing enzyme.

In some additional embodiments, other domains are targeted for amutation which results in reducing catalytic efficiency of theendogenous glucose and/or cellobiose oxidizing enzyme. For example, insome embodiments, a mutation to one or more residues in a heme bindingdomain of a glucose and/or cellobiose oxidizing enzyme can result inreduced catalytic efficiency (See e.g., Rotsaert et al., Arch. Biochem.Biophys., 390:206-14 [2001]).

Similarly, in some embodiments, the genetic modification is aconditional mutation to a glucose and/or cellobiose oxidizing enzyme. Insome embodiments, the glucose and/or cellobiose oxidizing enzyme has atemperature sensitive mutation that renders the protein non-functional(i.e., inactive or less active) at (e.g. warm temperatures, such as37-42° C.), and functional (i.e., active) at colder temperatures.

Fungal Cells

In some embodiments, the present invention provides a fungal cell thatis a Basidiomycete belonging to the class Agaricomycetes or anAscomycete belonging to the subdivision Pezizomycotina that has beengenetically modified to reduce the amount of endogenous glucose and/orcellobiose oxidizing enzyme activity that is secreted by the cell, wherethe fungal cell is capable of secreting a cellulase-containing enzymemixture. In some embodiments, the genetically modified fungal cellprovided herein is a Basidiomycete belonging to the class Agaricomycetesor an Ascomycete belonging to the subdivision Pezizomycotina. In someembodiments, the Basidiomycete is a species of Pleurotus, Peniophora,Trametes, Athelia, Sclerotium, Termitomyces, Flammulina, Coniphora,Ganoderma, Pycnoporus, Ceriporiopsis, Phanerochaete, Gloeophyllum,Hericium, Heterobasidion, Gelatoporia, Lepiota, or Irpex. In someembodiments, the Ascomycete is a species of Myceliophthora, Thielavia,Sporotrichum, Neurospora, Sordaria, Podospora, Magnaporthe, Fusarium,Gibberella, Botryotinia, Humicola, Neosartorya, Pyrenophora,Phaeosphaeria, Sclerotinia, Chaetomium, Nectria, Verticillium, orAspergillus.

The classification of a given fungal cell as belonging to theBasidiomycete class Agaricomycetes or to the Ascomycetes subdivisionPezizomycotina is done as is recognized in the art, as exemplified inthe NCBI taxonomy database.

In some embodiments, the fungal cell is a Chaetomiaceae family member.The Chaetomiaceae are a family of fungi in the Ascomycota, classSordariomycetes. The family Chaetomiaceae includes the generaAchaetomium, Aporothielavia, Chaetomidium, Chaetomium, Corylomyces,Corynascus, Farrowia, Thielavia, Zopfiella, and Myceliophthora.

In some embodiments, the genetically modified fungal cell is an anamorphor teleomorph of a Basidiomycete belonging to the class Agaricomycetesor an Ascomycete belonging to the subdivision Pezizomycotina. In someembodiments, the genetically modified fungal cell is an anamorph orteleomorph of a Chaetomiaceae family member selected from the generaMyceliophthora, Thielavia, Corynascus, Chaetomium. As such, thegenetically modified fungal cell can also be selected from the generaSporotrichum, Chrysosporium, Paecilomyces, Talaromyces or Acremonium. Itis also contemplated that the genetically modified fungal cell beselected from the genera Ctenomyces, Thermoascus, and Scytalidium,including anamorphs and teleomorphs of fungal cells from those genera.

In some further embodiments, the genetically modified fungal cell is athermophilic member of the genera Acremonium, Arthroderma, Corynascus,Thielavia, Myceliophthora, Thermoascus, Chromocleista, Byssochlamys,Sporotrichum, Chaetomium, Chrysosporium, Scytalidium, Ctenomyces,Paecilomyces, and Talaromyces. By “thermophilic fungus” is meant anyfungus which exhibits optimum growth at a temperature of at least about37° C., and generally below about 80° C., such as for example betweenabout 37-80° C., also between about 37-75° C., also between about 40-65°C., and also between about 40-60° C. In some embodiments, the optimumgrowth is exhibited at a temperature of at least 40°-60° C.

In some embodiments, the genetically modified fungal cell is selectedfrom the strains of Sporotrichum cellulophilum, Thielaviaheterothallica, Corynascus heterothallicus, Thielavia terrestris, andMyceliophthora thermophila, including anamorphs and teleomorphs thereof.It will be understood that for the aforementioned species, thegenetically modified fungal cell presented herein encompasses both theperfect and imperfect states, and other taxonomic equivalents (e.g.,anamorphs), regardless of the species name by which they are known. Forexample, the following species are anamorphs or teleomorphs and maytherefore be considered as synonymous: Myceliophthora thermophila,Sporotrichum thermophile, Sporotrichum thermophilum, Sporotrichumcellulophilum, Chrysosporium thermophile, Corynascus heterothallicus,and Thielavia heterothallica. Additionally, the following species may beconsidered synonymous with each other: Thielavia terrestris, Allscheriaterrestris, and Acremonium alabamense. Further examples of taxonomicequivalents are known in the art (See e.g., Cannon, Mycopathol.,111:75-83 [1990]; Moustafa et al., Persoonia 14:173-175 [1990];Stalpers, Stud. Mycol., 24, [1984]; Upadhyay et al., Mycopathol.,87:71-80 [1984]; Guarro et al., Mycotaxon 23: 419-427 [1985]; Awao etal., Mycotaxon 16:436-440 [1983]; von Klopotek, Arch. Microbiol.,98:365-369 [1974]; and Long et al., ATCC Names of Industrial Fungi,ATCC, Rockville Md. [1994]). Those skilled in the art will readilyrecognize the identity of appropriate equivalents. Accordingly, it willbe understood that, unless otherwise stated, the use of a particularspecies designation in the present disclosure also refers to speciesthat are related by anamorphic or teleomorphic relationship.

In some embodiments, the genetically modified fungal cell is acellulase-producing fungal cell that is a Basidiomycete belonging to theclass Agaricomycetes or an Ascomycete belonging to the subdivisionPezizomycotina. For example, in some embodiments, the geneticallymodified fungal cell is a Basidiomycete belonging to the classAgaricomycetes or an Ascomycete belonging to the subdivisionPezizomycotina that secretes two or more cellulose hydrolyzing enzymes,such as, for example, endoglucanase, cellobiohydrolase, orbeta-glucosidase, It will be appreciated that cellulase can includehemicellulose-hydrolyzing enzymes such as endoxylanase, beta-xylosidase,arabinofuranosidase, alpha-glucuronidase, acetylxylan esterase, feruloylesterase, and alpha-glucuronyl esterase. It will also be appreciatedthat a cellulase-producing fungal cell can produce two or more of theseenzymes, in any combination.

Additionally, in some embodiments, the genetically modified fungal cellis derived from a lignocellulose-competent parental fungal cell Thepresent invention also provides a fungal culture in a vessel comprisinga genetically modified fungal cell as described hereinabove. In someembodiments, the vessel comprises a liquid medium, such as fermentationmedium. For example, the vessel can be a flask, bioprocess reactor, andthe like. In some embodiments, the vessel comprises a solid growthmedium. For example, the solid medium can be an agar medium such aspotato dextrose agar, carboxymethylcellulose, cornmeal agar, and thelike. In some embodiments, the fungal cell described herein is anisolated fungal cell.

Sugar Oxidizing Enzymes

The present invention provides fungal cells that have been geneticallymodified to reduce the amount of endogenous glucose and/or cellobioseoxidizing enzyme secreted by the fungal cell. Examples of some suitableglucose and/or cellobiose oxidizing enzymes that find use in the presentinvention are described in greater detail below.

Glucose Oxidase. As indicated herein, in some embodiments, glucoseoxidase and fungal cells producing reduced levels of glucose oxidaseactivity find use in the present invention. Glucose oxidase is known tofunction via a so-called ping-pong mechanism of enzymatic catalysis,which involves successive binding on two different sites. One site is asaccharide binding domain that is capable of binding β-D-glucose. Theother site is a relatively non-selective co-substrate site for bindingan oxidant such as FAD.

One of skill in the art will appreciate that glucose oxidase enzymeactivity typically employs the presence of oxygen or an equivalent redoxacceptor (e.g., lignin, molecular oxygen, cytochrome c, redox dyes,benzoquinones and Fe²⁺ complexes). Glucose oxidase (GO) activity can bemeasured using any of a variety of methods known in the art. Forexample, GO activity assays can be performed using any suitable methodknown in the art (See e.g., Bergmeyer et al., in Methods of EnzymaticAnalysis (Bergmeyer, ed.) Volume 1, 2^(nd) Ed., pp. 457-458, AcademicPress Inc., New York, N.Y. [1974]; and U.S. Pat. No. 3,953,295). Forexample, GO activity is determined by an increase in absorbance at 460nm resulting from the oxidation of o-dianisidine through a peroxidasecoupled system.

In some embodiments, the present invention provides fungal cells thathave been genetically modified to reduce the secreted activity of aglucose oxidase and have reduced secreted activity of an endogenousglucose oxidase. Accordingly, one or more glucose oxidase enzymes fromeach of the fungal species described herein can be targeted for geneticmodification.

In some embodiments, the glucose oxidase is from a fungal species fromthe division Basidiomycete and belonging to the class Agaricomycetes; orfrom the division Ascomycete and belonging to the subdivisionPezizomycotina. Some examples of glucose oxidase enzymes identified fromdivision Basidiomycete belonging to the class Agaricomycetes; anddivision Ascomycete belonging to the subdivision Pezizomycotina are setforth in the Table B, below.

In some embodiments, the glucose oxidase is from a fungal speciesselected from Chaetomium globosum, Thielavia heterothallica, Thielaviaterrestris, Talaromyces stipitatus and Myceliophthora thermophila. Someglucose oxidase enzymes identified from these and other species are setforth in Table B, below. The proteins listed in Table B are examples ofglucose oxidase that are known in the art, or identified herein as beinga glucose oxidase.

TABLE B Glucose Oxidase Sequences GMC GMC oxred N oxred C AccessionNumber Organism Domain Domain chr1-56652m21GM Myceliophthora thermophila36-356 495-634 (SEQ ID NO.: 2) XP_001227361.1 Chaetomium globosum CBS148.51 36-355 465-604 JGIThite5217 Thielavia terrestris 36-355 466-605|XP_001223540.1 Chaetomium globosum CBS 148.51 185-380  514-655XP_001910674.1 Podospora anserina S mat+ 39-373 506-644 XP_001220376.1Chaetomium globosum CBS 148.51 38-342 481-620 XP_001226009.1 Chaetomiumglobosum CBS 148.51 214-289  323-464 CBI59558.1 Sordaria macrospora43-373 480-619 XP_383916.1 Gibberella zeae PH-1 35-345 466-603CBI59559.1 Sordaria macrospora 43-356 440-579 JGIThite6377 Thielaviaterrestris 40-363 490-630 XP_001549389.1 Botryotinia fuckeliana B05.1039-347 438-578 XP_001903685.1 Podospora anserina S mat+ 36-337 465-606XP_002792207.1 Paracoccidioides brasiliensis Pb01 68-290 414-556XP_003001656.1 Verticillium albo-atrum VaMs.102 46-359 488-627XP_361250.1 Magnaporthe oryzae 70-15 113-324  450-594 JGIThite5048Thielavia terrestris 45-354 480-619 XP_001226113.1 Chaetomium globosumCBS 148.51 44-353 480-619 XP_001906345.1 Podospora anserina S mat+39-352 480-620 chr4-293m24GM Myceliophthora thermophila 81-380 508-647(SEQ ID NO.: 4) CAJ85791.1 Fusarium oxysporum f. sp. Lycopersici 34-344465-602 XP_661610.1 Aspergillus nidulans FGSC A4 35-346 467-604XP_003041895.1 Nectria haematococca mpVI 77-13-4 35-346 467-604XP_001912227.1 Podospora anserina S mat+ 27-333 443-584 XP_681267.1Aspergillus nidulans FGSC A4 38-343 471-609 XP_001227424.1 Chaetomiumglobosum CBS 148.51 42-374 500-640 CBI58590.1 Sordaria macrospora 46-351477-588 EEH49925.1 Paracoccidioides brasiliensis Pb18 176-397  NAXP_001223186.1 Chaetomium globosum CBS 148.51 37-292 424-599XP_001791201.1 Phaeosphaeria nodorum SN15 60-223 349-480 XP_001905375.1Podospora anserina S mat+ 49-370 495-633 XP_001592050.1 Sclerotiniasclerotiorum 1980 227-296  314-446 XP_003002940.1 Verticilliumalbo-atrum VaMs.102 43-355 462-600 XP_366260.2 Magnaporthe oryzae 70-1542-351 491-631 XP_003048882.1 Nectria haematococca mpVI 77-13-4 121-295 394-530 XP_001796868.1 Phaeosphaeria nodorum SN15 38-350 475-615XP_001805358.1 Phaeosphaeria nodorum SN15 20-328 454-593 XP_001931252.1Pyrenophora tritici-repentis Pt-1C-BFP 23-318 508-595 XP_001218113.1Aspergillus terreus NIH2624 43-354 475-612 XP_001224467.1 Chaetomiumglobosum CBS 148.51 218-318  446-552 XP_003049247.1 Nectria haematococcampVI 77-13-4 25-323 451-589 XP_001906627.1 Podospora anserina S mat+37-349 473-618 XP_001804484.1 Phaeosphaeria nodorum SN15 29-319 449-586JGIThite9772 Thielavia terrestris 33-357 460-599 EEH08138.1 Ajellomycescapsulatus G186AR 181-256  366-509 XP_002565293.1 Penicilliumchrysogenum Wisconsin 54- 34-355 458-595 1255 CBI52485.1 Sordariamacrospora 28-338 NA XP_001273036.1 Aspergillus clavatus NRRL 1 14-322421-560 AAF31169.1|AF143814_1 Pleurotus pulmonarius 30-342 446-585EER39780.1 Ajellomyces capsulatus H143 228-301  411-554 XP_001558188.1Botryotinia fuckeliana B05.10 37-358 482-619 XP_001904543.1 Podosporaanserina S mat+ 27-344 453-596 XP_001544530.1 Ajellomyces capsulatusNAm1 228-301  411-554 JGIThite8281 Thielavia terrestris  5-301 456-624XP_383802.1 Gibberella zeae PH-1 24-324 452-589 XP_001833868.1Coprinopsis cinerea okayama7#130 35-341 430-568 XP_001836103.1Coprinopsis cinerea okayama7#130 39-366 462-535 XP_001597615.1Sclerotinia sclerotiorum 1980 134-290  295-422 ADD14021.1 Pleurotuseryngii 30-342 446-585 EEH50230.1 Paracoccidioides brasiliensis Pb18131-252  366-484 JGIThite6435 Thielavia terrestris 100-404  509-658XP_362999.2 Magnaporthe oryzae 70-15 31-327 431-575 XP_761104.1 Ustilagomaydis 521 54-372 485-603 XP_002482522.1 Talaromyces stipitatus ATCC10500 36-362 456-593 XP_001879270.1 Laccaria bicolor S238N-H82 35-345451-581 XP_001584680.1 Sclerotinia sclerotiorum 1980 24-335 440-581XP_001798598.1 Phaeosphaeria nodorum SN15 35-337 440-584 XP_661816.1Aspergillus nidulans FGSC A4 33-350 461-599 XP_001883085.1 Laccariabicolor S238N-H82 34-348 450-594 XP_001556658.1 Botryotinia fuckelianaB05.10 39-364 464-603 XP_001833865.1 Coprinopsis cinerea okayama7#13034-345 432-570 XP_002390024.1 Moniliophthora perniciosa FA553 36-298411-536 XP_001801353.1 Phaeosphaeria nodorum SN15 22-332 436-579XP_001817515.1 Aspergillus oryzae RIB40 28-335 436-577 XP_568317.1Cryptococcus neoformans var. 55-373 507-643 neoformans JEC21XP_001273087.1 Aspergillus clavatus NRRL 1 51-369 481-620 XP_002372599.1Aspergillus flavus NRRL3357 28-340 441-582 XP_001806098.1 Phaeosphaerianodorum SN15 62-382 494-634 XP_001835456.1 Coprinopsis cinereaokayama7#130 34-345 433-569 XP_001586361.1 Sclerotinia sclerotiorum 198032-346 446-586 XP_001884302.1 Laccaria bicolor S238N-H82 36-348 452-589XP_001821530.1 Aspergillus oryzae RIB40 75-394 496-635 XP_760191.1Ustilago maydis 521 59-366 497-628 XP_002375018.1 Aspergillus flavusNRRL3357 53-358 466-631 XP_391162.1 Gibberella zeae PH-1 21-316 442-579XP_759762.1 Ustilago maydis 521 84-408 530-667 CBI51995.1 Sordariamacrospora 30-326 469-610 XP_002376612.1 Aspergillus flavus NRRL335722-321 457-599 XP_660833.1 Aspergillus nidulans FGSC A4 41-358 466-603EDP53840.1 Aspergillus fumigatus A1163 36-347 448-589 XP_001911514.1Podospora anserina S mat+ 77-396 498-637 XP_002471526.1 Postia placentaMad-698-R 86-400 517-660 XP_367669.2 Magnaporthe oryzae 70-15 21-323427-568 XP_001389920.1 Aspergillus niger 5-289 391-528 XP_001732158.1Malassezia globosa CBS 7966 52-370 481-618 XP_001826806.1 Aspergillusoryzae RIB40 27-338 439-580 XP_001216916.1 Aspergillus terreus NIH262426-338 439-580 XP_003000545.1 Verticillium albo-atrum VaMs.102 21-339440-581 XP_391184.1 Gibberella zeae PH-1 264-389  491-631 XP_002148263.1Penicillium marneffei ATCC 18224 30-351 469-606 XP_749312.1 Aspergillusfumigatus Af293 36-347 448-589 JGIThite9811 Thielavia terrestris 22-337440-579 XP_001907031.1 Podospora anserina S mat+ 29-322 451-590XP_001550244.1 Botryotinia fuckeliana B05.10 24-336 451-484XP_002479433.1 Talaromyces stipitatus ATCC 10500 42-367 467-606XP_001390806.1 Aspergillus niger 38-356 466-605 XP_681081.1 Aspergillusnidulans FGSC A4 143-367  469-608 XP_001882478.1 Laccaria bicolorS238N-H82 33-345 451-588 XP_664049.1 Aspergillus nidulans FGSC A4 36-353464-603 XP_002373928.1 Aspergillus flavus NRRL3357 41-353 465-604XP_001592756.1 Sclerotinia sclerotiorum 1980 42-358 469-539 YP_914851.1Paracoccus denitrificans PD1222 13-304 394-530 XP_002622246.1Ajellomyces dermatitidis SLH14081 39-349 459-602 XP_002565328.1Penicillium chrysogenum Wisconsin 54- 34-338 450-589 1255 XP_001215452.1Aspergillus terreus NIH2624 46-350 454-589 XP_001820476.1 Aspergillusoryzae RIB40 41-343 455-594 XP_001732090.1 Malassezia globosa CBS 796655-373 484-621 XP_002388554.1 Moniliophthora perniciosa FA553 46-367487-624 XP_001265740.1 Neosartorya fischeri NRRL 181 34-345 446-587XP_381957.1 Gibberella zeae PH-1 53-320 432-553 XP_001587168.1Sclerotinia sclerotiorum 1980 33-351 464-603 XP_660308.1 Aspergillusnidulans FGSC A4 27-334 437-568 YP_001923964.1 Methylobacterium populiBJ001 67-360 428-563 XP_001559357.1 Botryotinia fuckeliana B05.10 50-370482-621 XP_002389049.1 Moniliophthora perniciosa FA553 33-345 NAXP_001394544.1 Aspergillus niger 27-338 439-580 XP_760250.1 Ustilagomaydis 521 38-293 419-556 XP_359722.1 Magnaporthe oryzae 70-15 39-365476-616 XP_001800211.1 Phaeosphaeria nodorum SN15 122-375  485-623XP_002481914.1 Talaromyces stipitatus ATCC 10500  5-303 416-553XP_001398576.1 Aspergillus niger 40-353 465-604 XP_003040786.1 Nectriahaematococca mpVI 77-13-4 61-379 493-631 XP_001904483.1 Podosporaanserina S mat+ 120-397  510-649 XP_759393.1 Ustilago maydis 521 40-366478-618 XP_002388140.1 Moniliophthora perniciosa FA553 25-346 NAXP_002373140.1 Aspergillus flavus NRRL3357 43-357 469-603 XP_002143250.1Penicillium marneffei ATCC 18224 37-355 466-605 XP_001729093.1Malassezia globosa CBS 7966 35-351 454-604 XP_001793977.1 Phaeosphaerianodorum SN15 25-345 451-587 XP_002476910.1 Postia placenta Mad-698-R 5-307 431-563 XP_001559633.1 Botryotinia fuckeliana B05.10 33-352465-604 XP_001732157.1 Malassezia globosa CBS 7966 49-368 480-617XP_002149622.1 Penicillium marneffei ATCC 18224 43-324 400-532XP_001910399.1 Podospora anserina S mat+ 51-369 480-620 XP_391404.1Gibberella zeae PH-1 64-382 496-634 XP_002794971.1 Paracoccidioidesbrasiliensis Pb01  5-300 414-551 XP_001270826.1 Aspergillus clavatusNRRL 1 48-372 484-623 XP_001548196.1 Botryotinia fuckeliana B05.1041-349 477-617 XP_001215424.1 Aspergillus terreus NIH2624 41-351 450-591XP_758019.1 Ustilago maydis 521 36-343 457-608 BAI66412.1 Fusariumoxysporum 66-382 496-634 EDP52931.1 Aspergillus fumigatus A1163 46-370482-621 XP_002387978.1 Moniliophthora perniciosa FA553 224-327  NAXP_001216137.1 Aspergillus terreus NIH2624 27-340 441-525 XP_002375706.1Aspergillus flavus NRRL3357 51-359 471-608 XP_001598641.1 Sclerotiniasclerotiorum 1980 50-370 482-621 gi|71002308|ref|XP_755835.1 Aspergillusfumigatus Af293 38-354 466-602 gi|119481873|ref|XP_001260965.1Neosartorya fischeri NRRL 181 38-354 466-605 XP_001817967.1 Aspergillusoryzae RIB40 43-357 469-608 XP_001275639.1 Aspergillus clavatus NRRL 139-356 468-607 XP_002148584.1 Penicillium marneffei ATCC 18224 54-377490-629 XP_002485672.1 Talaromyces stipitatus ATCC 10500 54-377 476-605EDP55006.1 Aspergillus fumigatus A1163 38-354 466-602 XP_754807.1Aspergillus fumigatus Af293 46-370 482-621 CBF78527.1 Aspergillusnidulans FGSC A4 21-324 436-575 XP_002556907.1 Penicillium chrysogenumWisconsin 54- 28-339 NA 1255 XP_002567445.1 Penicillium chrysogenumWisconsin 54- 53-377 485-622 1255 XP_001396848.1 Aspergillus niger60-366 454-593 XP_001397016.1 Aspergillus niger 51-381 502-641XP_001023507.1 Tetrahymena thermophila  8-309 403-539 XP_001263633.1Neosartorya fischeri NRRL 181 46-370 482-621 XP_001400283.1 Aspergillusniger 49-372 477-616 XP_001261659.1 Neosartorya fischeri NRRL 181 44-369477-612 XP_001797048.1 Phaeosphaeria nodorum SN15 39-366 458-598XP_001211074.1 Aspergillus terreus NIH2624 44-320 430-565 XP_001398522.1Aspergillus niger 50-373 475-614 *Accession numbers for Thielaviaterrestris refer to the U.S. Department of Energy (DOE) Joint GenomeInstitute (JGI) genome sequence

Some amino acid sequences encoding glucose oxidase are provided herein.For example, the nucleotide sequence encoding Myceliophthora thermophilaGO1 is set forth herein as SEQ ID NO:1, and the encoded amino acidsequence of Myceliophthora thermophila GO1 is set forth as SEQ ID NO:2.Furthermore, the nucleotide sequence encoding Myceliophthora thermophilaGO2 is set forth herein as SEQ ID NO:3, and the encoded amino acidsequence of Myceliophthora thermophila GO2 is set forth as SEQ ID NO:4.

In some embodiments, the glucose oxidase is glucose oxidase (EC1.1.3.4). In some embodiments, the glucose oxidase is a glucose oxidasewith the amino acid sequence of Myceliophthora thermophila GO1 as setforth in SEQ ID NO:2. In some embodiments, the glucose oxidase is aglucose oxidase with the amino acid sequence of Myceliophthorathermophila GO2 as set forth in SEQ ID NO:4. In other embodiments, theglucose oxidase comprises an amino acid sequence provided in the GenBankentry of any one of the accession numbers set forth in Table B. In someembodiments, the glucose oxidase is encoded by a nucleic acid sequencethat is at least about 60%, about 61%, about 62%, about 63%, about 64%,about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%,about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, or about 100% identical to SEQ ID NOS:1and/or 3. In some embodiments, the glucose oxidase is encoded by anucleic acid sequence that is at least about 60%, about 61%, about 62%,about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%,about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99%, or about 100% identicalto a nucleic acid sequence encoding the amino acid sequence set forth asSEQ ID NOS:2 and/or 4, or an amino acid sequence provided in the GenBankentry of any one of the accession numbers set forth in Table B. In someembodiments, the glucose oxidase is encoded by a nucleic acid sequencethat can selectively hybridize to SEQ ID NOS:1 and/or 3 under moderatelystringent or stringent conditions, as described below. In someembodiments, the glucose oxidase is encoded by a nucleic acid sequencethat can selectively hybridize under moderately stringent or stringentconditions to a nucleic acid sequence that encodes SEQ ID NOS:2 and/or4, or an amino acid sequence provided in the GenBank entry of any one ofthe accession numbers set forth in Table B.

In some embodiments, the glucose oxidase comprises an amino acidsequence with at least about 50%, about 51%, about 52%, about 53%, about54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%,about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%,about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or about 100% similarity to the amino acid sequence set forth as SEQ IDNOS:2 and/or 4, or an amino acid sequence provided in the GenBank entryof any one of the accession numbers set forth in Table B.

Cellobiose Dehydrogenase. In some embodiments, the CDH contains both theconserved glucose-methanol-choline (GMC) oxido-reductase N and the GMCoxido-reductase C domains. In some other embodiments, a CDH contains theGMC oxido-reductase N domain alone. The GMC oxidoreductases are FADflavoproteins oxidoreductases (See e.g., Cavener, J. Mol. Biol.,223:811-814 [1992]; and Vrielink and Blow, Biochem., 32:11507-15[1993]). The GMC oxidoreductases include a variety of proteins; cholinedehydrogenase (CHD), methanol oxidase (MOX) and cellobiose dehydrogenase(CDH) which share a number of regions of sequence similarities. One ofthese regions, located in the N-terminal section, corresponds to the FADADP-binding domain, as further defined by the Pfam database under theentry GMC_oxred_N (PF00732). Similarly, the C-terminal conserved domain(GMC oxido-reductase C domain) is defined as set forth in the Pfamdatabase under the entry GMC_oxred_C (PF05199).

Cellobiose dehydrogenases can be categorized into two families, where afirst family contains a catalytic portion and a second family contains acatalytic portion and a cellulose binding motif (CBM). Thethree-dimensional structure of an example cellobiose dehydrogenasefeatures two globular domains, each containing one of two cofactors: aheme or a flavin. The active site lies at a cleft between the twodomains. Oxidation of cellobiose typically occurs via 2-electrontransfer from cellobiose to the flavin, generatingcellobiono-1,5-lactone and reduced flavin. Active FAD is regenerated byelectron transfer to the heme group, leaving a reduced heme. The nativestate heme is regenerated by reaction with the oxidizing substrate atthe second active site.

The acceptor is preferentially iron ferricyanide, cytochrome C, or anoxidized phenolic compound such as dichloroindophenol (DCIP), anacceptor commonly used for colorimetric assays. Metal ions and O₂ arealso acceptors, but for most cellobiose dehydrogenases the reaction rateof cellobiose oxidase for these acceptors is several orders of magnitudelower than that observed for iron or organic oxidants. Followingcellobionolactone release, the product may undergo spontaneousring-opening to generate cellobionic acid (Hallberg et al., 2003, J.Biol. Chem. 278: 7160-7166).

Those of skill in the art will appreciate that cellobiose dehydrogenaseenzyme activity typically employs the presence of oxygen or anequivalent redox acceptor (e.g., lignin, molecular oxygen, cytochrome c,redox dyes, benzoquinones and Fe²⁺ complexes).

Cellobiose dehydrogenase activity can be measured using any of a varietyof methods known in the art. For example, CDH activity assays can beperformed using any suitable method known in the art (See e.g., Schou etal., Biochem J., 220:565-71 [1998]). For example, DCPIP(2,6-dichlorophenolindophenol) reduction by CDH activity in the presenceof cellobiose can be monitored by absorbance at 530 nm.

In some embodiments, the fungal cells provided herein that have beengenetically modified to reduce the secreted activity of a cellobiosedehydrogenase have reduced secreted activity of an endogenous cellobiosedehydrogenase. Accordingly, one or more cellobiose dehydrogenase enzymesfrom each of the fungal species described herein can be targeted forgenetic modification.

In some embodiments, the cellobiose dehydrogenase is from a fungalspecies in the division Basidiomycete and belonging to the classAgaricomycetes; or in the division Ascomycete and belonging to thesubdivision Pezizomycotina. Some examples of cellobiose dehydrogenaseenzymes identified from division Basidiomycete belonging to the classAgaricomycetes; and division Ascomycete belonging to the subdivisionPezizomycotina are set forth in Table C, below.

In some embodiments, the cellobiose dehydrogenase is from a fungalspecies selected from Thielavia heterothallica, Thielavia terrestris,Chaetomium globosum and Myceliophthora thermophila. Some cellobiosedehydrogenase enzymes identified from these and other species are setforth in the table below. The proteins listed in Table C are examples ofcellobiose dehydrogenase that are known in the art, or identified hereinas being a cellobiose dehydrogenase.

TABLE C Cellobiose Dehydrogenase Sequences GMC oxred N GMC oxred CAccession Number Organism Domain Domain AC26221 Myceliophthorathermophila 251-554 645-781 (SEQ ID NO.: 6) AAC26221 Myceliophthorathermophila 251-554 645-781 ABS45566 Myriococcum thermophilum 251-554645-781 ABS45567 Myriococcum thermophilum 251-554 645-781 CHGT_03380Chaetomium globosum 226-529 620-757 XP_001229896.1 Chaetomium globosumCBS 226-529 620-757 148.51 JGIThite5441 Thielavia terrestris 253-555647-783 CAP68427 Podospora anserina 247-550 643-779 CBI53519.1 Sordariamacrospora 252-554 645-782 XP_956591.1 Neurospora crassa OR74A 253-555646-782 XP_360402.2 Magnaporthe oryzae 70-15 264-566 657-794 EDP55266Aspergillus fumigatus A1163 265-568 661-796 BAE61169 Aspergillus oryzae254-556 647-782 CBI59551.1 Sordaria macrospora 167-295 382-518 EAW14611Aspergillus clavatus NRRL 1 254-556 647-783 XP_001209295.1 Aspergillusterreus NIH2624 253-474 586-720 JGIThite4524 Thielavia terrestris 36-337 NA CHGT_08276 Chaetomium globosum  36-338 NA XP_001225932.1Chaetomium globosum CBS  36-338 NA 148.51 JGIThite6738 Thielaviaterrestris 249-550 642-779 CAP61651 Podospora anserina 254-555 647-783AAF69005 Humicola insolens 247-548 640-776 XP_389261.1 Gibberella zeaePH-1 213-516 607-743 XP_958234.1 Neurospora crassa OR74A 274-576 668-804CDH2 derived from a C1 strain Myceliophthora 249-550 NA (SEQ ID NO.: 8)thermophila CBI54739.1 Sordaria macrospora 272-574 666-802XP_001800470.1 Phaeosphaeria nodorum SN15  36-343 366-502 XP_001939778.1Pyrenophora tritici-repentis Pt- 247-550 640-776 1C-BFP CHGT_08622Chaetomium globosum 249-521 549-667 XP_001226549.1 Chaetomium globosumCBS 249-521 549-667 148.51 XP_001801490.1 Phaeosphaeria nodorum SN15245-543 633-769 XP_001553707.1 Botryotinia fuckeliana B05.10 265-570656-796 XP_002999803.1 Verticillium albo-atrum 393-534 590-681 VaMs.102XP_001591237.1 Sclerotinia sclerotiorum 1980 265-570 655-796XP_001273175.1 Aspergillus clavatus NRRL 1 244-543 627-752 XP_749254.1Aspergillus fumigatus Af293 247-546 637-755 ACF60617 Ceriporiopsissubvermispora 236-519 634-763 BAC20641 Grifola frondosa 230-509 628-757AAC50004 Trametes versicolor 230-512 628-757 BAD32781 Coniophora puteana236-519 634-763 XP_367658.1 Magnaporthe oryzae 70-15  39-334 426-551BAD36748 Irpex lacteus 239-516 637-766 AAB61455 Phanerochaetechrysosporium 235-517 633-762 CAA61359 Phanerochaete chrysosporium234-516 632-761 AAO32063 Trametes versicolor 230-512 628-757 AAO64483Athelia rolfsii 233-520 631-760 XP_001265679.1 Neosartorya fischeri NRRL181 247-546 639-755 AAC32197 Pycnoporus cinnabarinus 231-517 628-758XP_383093.1 Gibberella zeae PH-1 27-325 408-528 2118247A Phanerochaetechrysosporium 234-515 631-759 XP_001937164.1 Pyrenophoratritici-repentis Pt- 245-542 625-754 1C-BFP XP_001400060.1 Aspergillusniger  33-329 415-542 CAP85828 Penicillium chrysogenum  30-327 393-537Wisconsin 54-1255 XP_001803287.1 Phaeosphaeria nodorum SN15 243-516 NAXP_003006847.1 Verticillium albo-atrum  27-317 411-529 VaMs.102XP_001402432.1 Aspergillus niger CBS 513.88 245-495 598-726XP_003042062.1 Nectria haematococca mpVI 241-540 597-754 77-13-4XP_001210806.1 Aspergillus terreus NIH2624  32-329 413-541 XP_386159.1Gibberella zeae PH-1  25-323 406-530 BAE63115 Aspergillus oryzae  24-317401-527 XP_001559563.1 Botryotinia fuckeliana B05.10 232-534 620-746XP_003003908.1 Verticillium albo-atrum 232-507 NA VaMs.102XP_003042935.1 Nectria haematococca mpVI 233-531 614-739 77-13-4XP_383918.1 Gibberella zeae PH-1 234-533 615-740 XP_001793048.1Phaeosphaeria nodorum SN15  28-314 408-542 BAE79276 Fusarium oxysporumf. sp.  27-325 408-532 Lycopersici XP_364344.1 Magnaporthe oryzae 70-15 49-321 426-560 XP_001547235.1 Botryotinia fuckeliana B05.10  30-313422-548 XP_001593342.1 Sclerotinia sclerotiorum 1980 232-536 622-748XP_385048.1 Gibberella zeae PH-1 239-538 595-752 XP_362950.1 Magnaportheoryzae 70-15  30-327 415-537 XP_003052041.1 Nectria haematococca mpVI 32-332 415-539 77-13-4 XP_001940494.1 Pyrenophora tritici-repentis Pt- 90-271 363-479 1C-BFP *Accession numbers for Thielavia terrestris referto the U.S. Department of Energy (DOE) Joint Genome Institute (JGI)genome sequence

Some amino acid sequences encoding cellobiose dehydrogenase are providedherein. For example, the nucleotide sequence encoding Myceliophthorathermophila CDH1 is set forth herein as SEQ ID NO:5, and the encodedamino acid sequence of Myceliophthora thermophila CDH1 is set forth asSEQ ID NO:6. Furthermore, the nucleotide sequence encodingMyceliophthora thermophila CDH2 is set forth herein as SEQ ID NO:7, andthe encoded amino acid sequence of Myceliophthora thermophila CDH2 isset forth as SEQ ID NO:8.

In some embodiments, the cellobiose dehydrogenase is cellobiosedehydrogenase (EC 1.1.99.18). In some embodiments, the cellobiosedehydrogenase is a cellobiose dehydrogenase with the amino acid sequenceof Myceliophthora thermophila CDH1 as set forth in SEQ ID NO:6. In someembodiments, the cellobiose dehydrogenase is a cellobiose dehydrogenasewith the amino acid sequence of Myceliophthora thermophila CDH2 as setforth in SEQ ID NO:8. In other embodiments, the cellobiose dehydrogenasecomprises an amino acid sequence provided in the GenBank entry of anyone of the accession numbers set forth in Table C. In some embodiments,the cellobiose dehydrogenase is encoded by a nucleic acid sequence thatis at least about 60%, about 61%, about 62%, about 63%, about 64%, about65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%,about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%,about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or about 100% identical to SEQ ID NOS:5 and/or 7.In some embodiments, the cellobiose dehydrogenase is encoded by anucleic acid sequence that is at least about 60%, about 61%, about 62%,about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%,about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99%, or about 100% identicalto a nucleic acid sequence encoding the amino acid sequence set forth asSEQ ID NOS:6 and/or 8, or an amino acid sequence provided in the GenBankentry of any one of the accession numbers set forth in Table D. In someembodiments, the cellobiose dehydrogenase is encoded by a nucleic acidsequence that can selectively hybridize to SEQ ID NOS:5 and/or 7 undermoderately stringent or stringent conditions, as described hereinabove.In some embodiments, the cellobiose dehydrogenase is encoded by anucleic acid sequence that can selectively hybridize under moderatelystringent or stringent conditions to a nucleic acid sequence thatencodes SEQ ID NOS:6 and/or 8, or an amino acid sequence provided in theGenBank entry of any one of the accession numbers set forth in Table C.

In some embodiments, the cellobiose dehydrogenase comprises an aminoacid sequence with at least about 50%, about 51%, about 52%, about 53%,about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%,about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%,about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about99%, or about 100% similarity to the amino acid sequence set forth asSEQ ID NOS:6 and/or 8, or an amino acid sequence provided in the GenBankentry of any one of the accession numbers set forth in Table C.Similarity as used herein is described in greater detail hereinabove.

Cellobiose dehydrogenase sequences can be identified by any of a varietyof methods known in the art. For example, a sequence alignment can beconducted against a database, for example against the NCBI database, andsequences with the lowest HMM E-value can be selected.

Pyranose Oxidase. As indicated herein, pyranose oxidases and fungalcells that have been modified to have reduced pyranose oxidase activityfind use in the present invention. Pyranose oxidase activity can bemeasured using any of a variety of methods known in the art. Forexample, PO activity assays can be performed as described by Leitner etal., Appl Biochem Biotechnol 1998, 70-72:237-248), which is incorporatedby reference in its entirety. For example,2′-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) reduction by POactivity can be monitored by absorbance at 530 nm. In some additionalembodiments, PO activity is determined by an increase in absorbance at420 nm resulting from the oxidation of2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) through aperoxidase coupled system.

In some embodiments, the fungal cells provided herein that have beengenetically modified to reduce the secreted activity of a pyranoseoxidase have reduced secreted activity of an endogenous pyranoseoxidase. Accordingly, one or more pyranose oxidase enzymes from each ofthe fungal species described herein can be targeted for geneticmodification.

In some embodiments, the pyranose oxidase is from a fungal species inthe division Basidiomycete and belonging to the class Agaricomycetes; orin the division Ascomycete and belonging to the subdivisionPezizomycotina. Some examples of pyranose oxidase enzymes identifiedfrom division Basidiomycete belonging to the class Agaricomycetes; anddivision Ascomycete belonging to the subdivision Pezizomycotina are setforth in Table D, below.

In some embodiments, the pyranose oxidase is from a fungal speciesselected from Peniophora gigantean, Phanerochaete chrysosporium,Trametes ochracea, Trametes pubescens, Emericella nidulans, Aspergillusoryzae, Gloeophyllum trabeum, Tricholoma matsutake, Trametes hirsute,Gloeophyllum trabeum, Phanerochaete chrysosporium, Peniophora sp.,Trametes versicolor, Lyophyllum shimeji, Trametes pubescens, Phlebiopsisgigantea, Aspergillus parasiticus, Auricularia polytricha, Coriolushirsutus, Coriolus versicolor, Gloeophyllum sepiarum, Iridophycusfaccidum, Irpex lactus, Oudemansiella mucida, Phanerochaete gigantea,Pleurotus ostreatus, Polyporus obtusus, Saxidomus giganteus, Todusmulticolor, Trametes cinnabarinus and Trametes multicolor. Some pyranoseoxidase enzymes identified from these species are set forth in Table D,below. The proteins listed in the table below are examples of pyranoseoxidase that are known in the art, or identified herein as being apyranose oxidase.

TABLE D Pyranose Oxidase Sequences Accession Database Number OrganismReference Swiss Prot Q6UG02 Peniophora gigantea Swiss Prot Q6QWR1Phanerochaete chrysosporium Swiss Prot Q7ZA32 Trametes ochracea SwissProt Q5G234 Trametes pubescens GenPept Q5B2E9 Emericella nidulansGenPept BAE56707.1 Aspergillus oryzae (SEQ ID NO: 10) GenPept ACJ54278.1Gloeophyllum trabeum GenPept BAC24805.1 Tricholoma matsutake GenPeptP59097 Trametes hirsuta GenPept ACM47528.1 Gloeophyllum trabeum GenPeptAAS93628.1 Phanerochaete chrysosporium GenPept AAO13382.1 Peniophora sp.GenPept BAA11119.1 Trametes versicolor GenPept BAD1079.1 Lyophyllumshimeji GenPept AAW57304.1 Trametes pubescens GenPept AAQ72486.1Phlebiopsis gigantea Aspergillus GiffhornAppl. Microbiol. Biotechnol.,54: 727-740 parasiticus (2000) Auricularia Izumi et al., Agric. Biol.Chem., 54: 799-801 polytricha (1990) Coriolus hirsutus Machida et al.,Agric. Biol. Chem.. 48: 2463-2470 (1984) Coriolus versicolor Taguchi etal., J. Appl. Biochem., 7: 289-295 (1985) Gloeophyllum Izumi et al.,Agric. Biol. Chem., 54: 799-801 sepiarum (1990) Iridophycus Giffhorn,Appl. Microbiol. Biotechnol., 54: 727-740 faccidum (2000) Irpex lactusIzumi et al., Agric. Biol. Chem., 54: 799-801 (1990) OudemansiellaGiffhorn, Appl. Microbiol. Biotechnol., 54: 727-740 mucida (2000)Phanerochaete Giffhorn, Appl. Microbiol. Biotechnol., 54: 727-740gigantean (2000) Pleurotus ostreatus Giffhorn, Appl. Microbiol.Biotechnol., 54: 727-740 (2000) Polyporus obtusus Janssen et al.,Methods Enzymol., 41B: 170-173) 1975_(—) Saxidomus giganteus Giffhorn,Appl. Microbiol. Biotechnol., 54: 727-740 (2000) Todus multicolorGiffhorn, Appl. Microbiol. Biotechnol., 54: 727-740 (2000) TrametesIzumi et al., Biol. Chem., 54: 799-801 (1990) cinnabarinus Trametesmulticolor Tasca et al., Electroanal., 19: 294-302 (2007)

Some amino acid sequences encoding pyranose oxidase are provided herein.For example, the nucleotide sequence encoding Aspergillus oryzae PO1 isset forth herein as SEQ ID NO:9, and the encoded amino acid sequence ofAspergillus oryzae PO1 is set forth as SEQ ID NO:10.

In some embodiments, the pyranose oxidase is pyranose oxidase (E.C.1.1.3.10). In some embodiments, the pyranose oxidase is a pyranoseoxidase with the amino acid sequence of Aspergillus oryzae PO1 as setforth in SEQ ID NO: 10. In other embodiments, the pyranose oxidasecomprises an amino acid sequence provided in the literature referenceand/or GenBank entry of any one of the accession numbers set forth inTable D. In some embodiments, the pyranose oxidase is encoded by anucleic acid sequence that is at least about 60%, about 61%, about 62%,about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%,about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99%, or about 100% identicalto SEQ ID NO:9. In some embodiments, the pyranose oxidase is encoded bya nucleic acid sequence that is at least about 60%, about 61%, about62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%,about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%,about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%identical to a nucleic acid sequence encoding the amino acid sequenceset forth as SEQ ID NO:10, or an amino acid sequence provided in theliterature reference and/or GenBank entry of any one of the accessionnumbers set forth in Table D. In some embodiments, the pyranose oxidaseis encoded by a nucleic acid sequence that can selectively hybridize toSEQ ID NO:9 under moderately stringent or stringent conditions, asdescribed hereinabove. In some embodiments, the pyranose oxidase isencoded by a nucleic acid sequence that can selectively hybridize undermoderately stringent or stringent conditions to a nucleic acid sequencethat encodes SEQ ID NO:10, or an amino acid sequence provided in theliterature reference and/or GenBank entry of any one of the accessionnumbers set forth in Table D.

In some embodiments, the pyranose oxidase comprises an amino acidsequence with at least about 50%, about 51%, about 52%, about 53%, about54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%,about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%,about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or about 100% similarity to the amino acid sequence set forth as SEQ IDNO:10, or an amino acid sequence provided in the literature referenceand/or GenBank entry of any one of the accession numbers set forth inTable D. Similarity as used herein is described in greater detailhereinabove.

Pyranose oxidase sequences can be identified by any of a variety ofmethods known in the art. For example, a sequence alignment can beconducted against a database, for example against the NCBI database, andsequences with the lowest HMM E-value can be selected.

Glucooligosaccharide Oxidase. As indicated herein, glucooligosaccharideoxidases and fungal cells modified to have reduced GOOX activity finduse in the present invention. Glucooligosaccharide oxidase activity canbe measured using any of a variety of methods known in the art. Forexample, GOOX activity assays can be performed as described by Lin etal., (Biochim. Biophys. Acta. 1991, 11:417-427), or Lee et al. (Appl.Environ. Microbiol. 2005, 71:8881-8887) each of which is incorporated byreference in its entirety. For example, activity can be measured bydetermining H₂O₂ production by coupling to a peroxidase enzyme assay.

In some embodiments, the fungal cells provided herein that have beengenetically modified to reduce the secreted activity of at least oneglucooligosaccharide oxidase have reduced secreted activity of anendogenous glucooligosaccharide oxidase. Accordingly, one or moreglucooligosaccharide oxidase enzymes from each of the fungal speciesdescribed herein can be targeted for genetic modification.

In some embodiments, the glucooligosaccharide oxidase is from a fungalspecies in the division Basidiomycete and belonging to the classAgaricomycetes; or in the division Ascomycete and belonging to thesubdivision Pezizomycotina. Some examples of glucooligosaccharideoxidase identified from division Basidiomycete belonging to the classAgaricomycetes; and division Ascomycete belonging to the subdivisionPezizomycotina are set forth in the table below.

In some embodiments, the glucooligosaccharide oxidase is from a fungalspecies selected from Acremonium strictum and Paraconiothyrium sp. Someglucooligosaccharide oxidase enzymes identified from these species areset forth in Table E, below. The proteins listed in the table below areexamples of glucooligosaccharide oxidase that are known in the art, oridentified herein as being a glucooligosaccharide oxidase.

TABLE E Glucooligosaccharide Oxidase Sequences Accession Database NumberOrganism Reference TrEMBL Q6PW77 Acremonium UniProt (SEQ ID strictum NO:12) Paraconiothyrium Kiryu et al., 2008, Biosci. sp. Biotechnol.Biochem., 72: 833-841 (2008)

Some amino acid sequences encoding glucooligosaccharide oxidase areprovided herein. For example, the nucleotide sequence encodingAcremonium strictum GOOX1 is set forth herein as SEQ ID NO:11, and theencoded amino acid sequence of Acremonium strictum GOOX1 is set forth asSEQ ID NO:12.

In some embodiments, the glucooligosaccharide oxidase isglucooligosaccharide oxidase (E.C. 1.1.99.B3). In some embodiments, theglucooligosaccharide oxidase is a glucooligosaccharide oxidase with theamino acid sequence of Acremonium strictum GOOX1 as set forth in SEQ IDNO:12. In other embodiments, the glucooligosaccharide oxidase comprisesan amino acid sequence provided in the literature reference and/orGenBank entry of any one of the accession numbers set forth in Table E.In some embodiments, the glucooligosaccharide oxidase is encoded by anucleic acid sequence that is at least about 60%, about 61%, about 62%,about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%,about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99%, or about 100% identicalto SEQ ID NO:11. In some embodiments, the glucooligosaccharide oxidaseis encoded by a nucleic acid sequence that is at least about 60%, about61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%,about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%,about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, orabout 100% identical to a nucleic acid sequence encoding the amino acidsequence set forth as SEQ ID NO:12, or an amino acid sequence providedin the literature reference and/or the GenBank entry of any one of theaccession numbers set forth in Table E. In some embodiments, theglucooligosaccharide oxidase is encoded by a nucleic acid sequence thatcan selectively hybridize to SEQ ID NO:11 under moderately stringent orstringent conditions, as described hereinabove. In some embodiments, theglucooligosaccharide oxidase is encoded by a nucleic acid sequence thatcan selectively hybridize under moderately stringent or stringentconditions to a nucleic acid sequence that encodes SEQ ID NO:12, or anamino acid sequence provided in the literature reference and/or GenBankentry of any one of the accession numbers set forth in Table E.

In some embodiments, the glucooligosaccharide oxidase comprises an aminoacid sequence with at least about 50%, about 51%, about 52%, about 53%,about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%,about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%,about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about99%, or about 100% similarity to the amino acid sequence set forth asSEQ ID NO:12, or an amino acid sequence provided in the literaturereference and/or the GenBank entry of any one of the accession numbersset forth in Table E. Similarity as used herein is described in greaterdetail hereinabove.

Glucooligosaccharide oxidase sequences can be identified by any of avariety of methods known in the art. For example, a sequence alignmentcan be conducted against a database, for example against the NCBIdatabase, and sequences with the lowest HMM E-value can be selected.

Pyranose Dehydrogenase. In some embodiments, pyranose dehydrogenases andfungal cells that have reduced pyranose dehydrogenase activity find usein the present invention. Pyranose dehydrogenases activity can bemeasured using any of a variety of methods known in the art. Forexample, PDH activity assays can be performed using any suitable method(See e.g., Volc et al., Arch. Microbiol., 176:178-186 [2001]).

In some embodiments, the fungal cells that have been geneticallymodified to reduce the secreted activity of a pyranose dehydrogenasehave reduced secreted activity of an endogenous pyranose dehydrogenase.Accordingly, one or more pyranose dehydrogenase enzymes from each of thefungal species described herein can be targeted for geneticmodification.

In some embodiments, the pyranose dehydrogenase is from a fungal speciesin the division Basidiomycete and belonging to the class Agaricomycetes;or in the division Ascomycete and belonging to the subdivisionPezizomycotina. Some examples of pyranose dehydrogenase identified fromdivision Basidiomycete belonging to the class Agaricomycetes; anddivision Ascomycete belonging to the subdivision Pezizomycotina are setforth in the table below.

In some embodiments, the pyranose dehydrogenase is from a fungal speciesselected from Agaricus bisporus, Agaricus meleagris, Agaricusxanthoderma, Macroleplota rhacodes and Leucoagaricus meleagris. Somepyranose dehydrogenase enzymes identified from these species are setforth in Table F, below. The proteins listed in the table below areexamples of pyranose dehydrogenase that are known in the art, oridentified herein as being a pyranose dehydrogenase.

TABLE F Pyranose Dehydrogenase Sequences Accession Database NumberOrganism Reference TrEMBL Q3L1D1 Agaricus bisporus UniProt (SEQ ID NO:14) TrEMBL Q3L245 Agaricus meleagris UniProt TrEMBL Q0R4L2 Agaricusmeleagris UniProt TrEMBL Q3L243 Agaricus meleagris UniProt TrEMBL Q3L1D2Agaricus UniProt xanthoderma Macroleplota Volc et al., Arch. rhacodesMicrobiol., 176: 178-186 (2001) GenBank AAW82996.1 Leucoagaricusmeleagris GenBank AAW82998.1 Leucoagaricus meleagris GenBank AAZ94874.1Leucoagaricus meleagris

Some amino acid sequences encoding pyranose dehydrogenase are providedherein. For example, the nucleotide sequence encoding Agaricus bisporusPDH1 is set forth herein as SEQ ID NO:13, and the encoded amino acidsequence of Agaricus bisporus PDH1 is set forth as SEQ ID NO:14.

In some embodiments, the pyranose dehydrogenase is pyranosedehydrogenase (E.C. 1.1.99.29). In some embodiments, the pyranosedehydrogenase is a pyranose dehydrogenase with the amino acid sequenceof Agaricus bisporus PDH1 as set forth in SEQ ID NO:14. In some otherembodiments, the pyranose dehydrogenase comprises an amino acid sequenceprovided in the literature reference and/or GenBank entry of any one ofthe accession numbers set forth in Table F. In some embodiments, thepyranose dehydrogenase is encoded by a nucleic acid sequence that is atleast about 60%, about 61%, about 62%, about 63%, about 64%, about 65%,about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%,about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99%, or about 100% identical to SEQ ID NO:13. In someembodiments, the pyranose dehydrogenase is encoded by a nucleic acidsequence that is at least about 60%, about 61%, about 62%, about 63%,about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%,about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or about 100% identical to anucleic acid sequence encoding the amino acid sequence set forth as SEQID NO:14, or an amino acid sequence provided in the literature referenceand/or GenBank entry of any one of the accession numbers set forth inTable F. In some embodiments, the pyranose dehydrogenase is encoded by anucleic acid sequence that can selectively hybridize to SEQ ID NO:13under moderately stringent or stringent conditions, as describedhereinabove. In some embodiments, the pyranose dehydrogenase is encodedby a nucleic acid sequence that can selectively hybridize undermoderately stringent or stringent conditions to a nucleic acid sequencethat encodes SEQ ID NO:14, or an amino acid sequence provided in theliterature reference and/or GenBank entry of any one of the accessionnumbers set forth in Table F.

In some embodiments, the pyranose dehydrogenase comprises an amino acidsequence with at least about 50%, about 51%, about 52%, about 53%, about54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%,about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%,about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or about 100% similarity to the amino acid sequence set forth as SEQ IDNO:14, or an amino acid sequence provided in the literature referenceand/or the GenBank entry of any one of the accession numbers set forthin Table F. Similarity as used herein is described in greater detailhereinabove.

Pyranose dehydrogenase sequences can be identified by any of a varietyof methods known in the art. For example, a sequence alignment can beconducted against a database, for example against the NCBI database, andsequences with the lowest HMM E-value can be selected.

Glucose Dehydrogenase. As indicated herein, glucose dehydrogenases andfungal cells that have been modified to have reduced glucosedehydrogenase activity find use in the present invention. Glucosedehydrogenase activity can be measured using any of a variety of methodsknown in the art (See e.g., Strecker, Meth. Enzymol., 1:335 [1955]). Insome embodiments, GDH activity is determined by an increase inabsorbance at 340 nm resulting from the generation of NADH from NAD whenbeta-D-glucose is provided as a substrate and NAD as an acceptor.

In some embodiments, the fungal cells provided herein that have beengenetically modified to reduce the secreted activity of a glucosedehydrogenase have reduced secreted activity of an endogenous glucosedehydrogenase. Accordingly, one or more glucose dehydrogenase enzymesfrom each of the fungal species described herein can be targeted forgenetic modification.

In some embodiments, the glucose dehydrogenase is from a fungal speciesin the division Basidiomycete and belonging to the class Agaricomycetes;or in the division Ascomycete and belonging to the subdivisionPezizomycotina. Some examples of glucose dehydrogenase identified fromdivision Basidiomycete belonging to the class Agaricomycetes; anddivision Ascomycete belonging to the subdivision Pezizomycotina are setforth in the table below.

In some embodiments, the glucose dehydrogenase is from a fungal speciesselected from Aspergillus niger, Aspergillus oryzae, Aspergillus terreusand Talaromyces stipatus. Some glucose dehydrogenase enzymes identifiedfrom these species are set forth in Table G, below. The proteins listedin the table below are examples of glucose dehydrogenase that are knownin the art, or identified herein as being a glucose dehydrogenase.

TABLE G Glucose Dehydrogenase Sequences Accession Database NumberOrganism Reference Aspergillus Muller, Zentralbl. niger Bacteriol.Parasienkd. Infectionskr Hyg., 132(a): 14-24 (1977) Aspergillus Bak,Biochim. oryzae Biophys. Acta 139: 277-293 (1967) Aspergillus Tsujimuraet al., terreus (2006) Biosci. Biotechnol. Biochem., 70: 654-659 (2006)GenBank XP_002482522.1 Talaromyces (SEQ ID NO: stipitatus ATCC 16) 10500GenBank XP_002479433.1 Talaromyces stipitatus ATCC 10500 GenBankXP_002481914.1 Talaromyces stipitatus ATCC 10500

Some amino acid sequences encoding glucose dehydrogenase are providedherein. For example, the nucleotide sequence encoding Myceliophthorathermophila GDH1 is set forth herein as SEQ ID NO:15, and the encodedamino acid sequence of Myceliophthora thermophila GDH1 is set forth asSEQ ID NO:16.

In some embodiments, the glucose dehydrogenase is glucose dehydrogenase(E.C. 1.1.99.10). In some embodiments, the glucose dehydrogenase is aglucose dehydrogenase with the amino acid sequence of Myceliophthorathermophila GDH1 as set forth in SEQ ID NO:16. In some otherembodiments, the glucose dehydrogenase comprises an amino acid sequenceprovided in the literature reference and/or GenBank entry of any one ofthe accession numbers set forth in Table G. In some embodiments, theglucose dehydrogenase is encoded by a nucleic acid sequence that is atleast about 60%, about 61%, about 62%, about 63%, about 64%, about 65%,about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%,about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99%, or about 100% identical to SEQ ID NO:15. In someembodiments, the glucose dehydrogenase is encoded by a nucleic acidsequence that is at least about 60%, about 61%, about 62%, about 63%,about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%,about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or about 100% identical to anucleic acid sequence encoding the amino acid sequence set forth as SEQID NO:16, or an amino acid sequence provided in the literature referenceand/or GenBank entry of any one of the accession numbers set forth inTable G. In some embodiments, the glucose dehydrogenase is encoded by anucleic acid sequence that can selectively hybridize to SEQ ID NO:15under moderately stringent or stringent conditions, as describedhereinabove. In some embodiments, the glucose dehydrogenase is encodedby a nucleic acid sequence that can selectively hybridize undermoderately stringent or stringent conditions to a nucleic acid sequencethat encodes SEQ ID NO:16, or an amino acid sequence provided in theliterature reference and/or GenBank entry of any one of the accessionnumbers set forth in Table G.

In some embodiments, the glucose dehydrogenase comprises an amino acidsequence with at least about 50%, about 51%, about 52%, about 53%, about54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%,about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%,about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or about 100% similarity to the amino acid sequence set forth as SEQ IDNO:16, or an amino acid sequence provided in the literature reference orthe GenBank entry of any one of the accession numbers set forth in TableG. Similarity as used herein is described in greater detail hereinabove.

Glucose dehydrogenase sequences can be identified by any of a variety ofmethods known in the art. For example, a sequence alignment can beconducted against a database, for example against the NCBI database, andsequences with the lowest HMM E-value selected, as desired.

In some embodiments, the cell has been genetically modified to reducethe amount of glucose and/or cellobiose oxidizing enzyme activity fromtwo or more endogenous glucose and/or cellobiose oxidizing enzymes thatare secreted by the cell. In certain such embodiments, a first of thetwo or more the glucose and/or cellobiose oxidizing enzymes comprises anamino acid sequence that is at least about 60%, about 61%, about 62%,about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%,about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99%, or about 100% identicalto any one of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14 and/or 16 and a secondof the two or more the glucose and/or cellobiose oxidizing enzymescomprises an amino acid sequence that is at least about 60%, about 61%,about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about100% identical to any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14 and/or16.

Enzyme Mixtures

Also provided herein are enzyme mixtures that comprise at least one ormore cellulose hydrolyzing enzymes expressed by a fungal cell that hasbeen genetically modified to reduce the amount of endogenous glucoseand/or cellobiose oxidizing enzyme activity that is secreted by thecell, as described hereinabove.

In some embodiments the enzyme mixture is in a vessel comprising agenetically modified fungal cell as described hereinabove. In someembodiments, the vessel comprises a liquid medium. For example, thevessel can be a flask, bioprocess reactor, and the like. In someembodiments, the enzyme mixture is in a liquid volume. For example, theliquid volume can be greater than about 0.01 mL, 0.1 mL, 1 mL, 10 mL,100 mL, 1000 mL, or greater than about 10 L, 50 L, 100 L, 200 L, 300 L,400 L, 500 L, 600 L, 700 L, 800 L, 900 L, 1000 L, 10,000 L, 50,000 L,100,000 L, 250,000 L, and 500,000 L or greater than about 1,000,000 L.

In some embodiments, the fungal cell is a lignocellulose-utilizing cellthat is a Basidiomycete belonging to the class Agaricomycetes or anAscomycete belonging to the subdivision Pezizomycotina, and where thefungal cell is capable of secreting a cellulase-containing enzymemixture. In some embodiments, the fungal cell is capable of secreting anenzyme mixture comprising two or more cellulase enzymes. In someembodiments, the Basidiomycete is a species of Pleurotus, Peniophora,Trametes, Athelia, Sclerotium, Termitomyces, Flammulina, Coniphora,Ganoderma, Pycnoporus, Ceriporiopsis, Phanerochaete, Gloeophyllum,Hericium, Heterobasidion, Gelatoporia, Lepiota, or Irpex. In someembodiments, the Ascomycete is a species of Myceliophthora, Thielavia,Sporotrichum, Neurospora, Sordaria, Podospora, Magnaporthe, Fusarium,Gibberella, Botryotinia, Humicola, Neosartorya, Pyrenophora,Phaeosphaeria, Sclerotinia, Chaetomium, Nectria, Verticillium, orAspergillus.

In some embodiments, the fungal cell is a lignocellulose-utilizing cellfrom the family Chaetomiaceae. In some embodiments, the geneticallymodified fungal cell provided herein is a Chaetomiaceae family memberselected from the genera Myceliophthora, Thielavia, Corynascus, orChaetomium. The genetically modified fungal cell can also be an anamorphor teleomorph of a Chaetomiaceae family member selected from the generaMyceliophthora, Thielavia, Corynascus, or Chaetomium. As such, thegenetically modified fungal cell can also be selected from the generaSporotrichum, Acremonium or Talaromyces. It is also contemplated thatthe genetically modified fungal cell be selected from the generaCtenomyces, Thermoascus, and Scytalidium, including anamorphs andteleomorphs of fungal cells from those genera. In some embodiments, thefungal cell is a species selected from Sporotrichum cellulophilum,Thielavia heterothallica, Corynascus heterothallicus, Thielaviaterrestris, Chaetomium globosum, Talaromyces stipitatus andMyceliophthora thermophila, including anamorphs and teleomorphs thereof.

In some embodiments, the present invention provides enzyme mixtures thatare cell-free. In some embodiments, two or more cellulases and anyadditional enzymes and/or other components present in the cellulaseenzyme mixtures are produced by a single type of genetically modifiedfungal cells, while in some embodiments the cellulases and/or otherenzymes and/or other components are produced by different microbes. Insome embodiments, the fermentations comprise single genetically modifiedcells and/or different microorganisms in combination, while in someother embodiments, the cells are grown in separate fermentations.Similarly, in some embodiments, the two or more cellulases and/or anyadditional enzymes and/or other components present in the cellulaseenzyme mixture are expressed individually or in sub-groups fromdifferent strains of different organisms and the enzymes combined invitro to produce the cellulase enzyme mixture. In some embodiments,cellulases and/or any additional enzymes and/or other components in theenzyme mixture are expressed individually or in sub-groups fromdifferent strains of a single organism, and the enzymes combined to makethe cellulase enzyme mixture. In some embodiments, all of the enzymesand/or other components are expressed from a single host organism, suchthe genetically modified fungal cell as describe herein above.

In some embodiments, the enzyme mixtures comprise at least one or morecellulose hydrolyzing enzymes expressed by a fungal cell that has beengenetically modified to reduce the amount of endogenous glucose and/orcellobiose oxidizing enzyme activity that is secreted by the cell, asdescribed hereinabove.

In some embodiments, the fungal cell is a lignocellulose-utilizing cellthat is a Basidiomycete belonging to the class Agaricomycetes or anAscomycete belonging to the subdivision Pezizomycotina, and where thefungal cell is capable of secreting a cellulase-containing enzymemixture. In some embodiments, the fungal cell is capable of secreting anenzyme mixture comprising two or more cellulase enzymes. In someembodiments, the Basidiomycete is a species of Pleurotus, Peniophora,Trametes, Athelia, Sclerotium, Termitomyces, Flammulina, Coniphora,Ganoderma, Pycnoporus, Ceriporiopsis, Phanerochaete, Gloeophyllum,Hericium, Heterobasidion, Gelatoporia, Lepiota, or Irpex. In someembodiments, the Ascomycete is a species of Myceliophthora, Thielavia,Sporotrichum, Neurospora, Sordaria, Podospora, Magnaporthe, Fusarium,Gibberella, Botryotinia, Humicola, Neosartorya, Pyrenophora,Phaeosphaeria, Sclerotinia, Chaetomium, Nectria, Verticillium, orAspergillus.

In some embodiments, the fungal cell is a lignocellulose-utilizing cellfrom the family Chaetomiaceae. In some embodiments, the geneticallymodified fungal cell provided herein is a Chaetomiaceae family memberselected from the genera Myceliophthora, Thielavia, Corynascus, andChaetomium. The genetically modified fungal cell can also be an anamorphor teleomorph of a Chaetomiaceae family member selected from the generaMyceliophthora, Thielavia, Corynascus, and Chaetomium. As such, thegenetically modified fungal cell can also be selected from the generaSporotrichum or Acremonium. It is also contemplated that the geneticallymodified fungal cell can also be selected from the genera Ctenomyces,Scytalidium and Thermoascus, including anamorphs and teleomorphs offungal cells from those genera. Typically, the fungal cell is a speciesselected from Sporotrichum cellulophilum, Thielavia heterothallica,Corynascus heterothallicus, Thielavia terrestris, Chaetomium globosum,Talaromyces stipitatus, and Myceliophthora thermophila, includinganamorphs and teleomorphs thereof.

Some cellulase mixtures for efficient enzymatic hydrolysis of cellulosethat are known (See e.g., Viikari et al., Adv. Biochem. Eng.Biotechnol., 108:121-45 [2007]; and US Pat. Appln. Publn. Nos. US2009/0061484, US 2008/0057541, and US 2009/0209009) find use ascomponents of some enzyme mixtures provided herein. In some embodiments,mixtures of purified naturally occurring or recombinant enzymes arecombined with cellulosic feedstock or a product of cellulose hydrolysis.Alternatively or in addition, one or more cell populations, eachproducing one or more naturally occurring or recombinant cellulases, arecombined with cellulosic feedstock or a product of cellulose hydrolysis.In some embodiments, the enzyme mixture comprises commercially availablepurified cellulases. Commercial cellulases are known and available tothe art. In some embodiments, the enzyme mixtures do not comprise anendoglucanase.

In some embodiments, the enzyme mixture comprises at least 5%, at least10%, at last 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, or at least 50% GH61. In someembodiments, the enzyme mixture further comprises a cellobiohydrolase 1a(e.g., CBH1a) and GH61, wherein the enzymes together comprise at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, or at least 80% of the enzyme mixture. In some embodiments, theenzyme mixture further comprises a β-gludosidase (Bgl), GH61, and CBH,wherein the three enzymes together comprise at least 30%, at least 35%,at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, or at least 85% ofthe enzyme mixture. In some embodiments, the enzyme mixture furthercomprises an endoglucanase (EG), GH61, CBH2b, CBH1a, Bgl, wherein thefive enzymes together comprise at least 35%, at least 40%, at least 45%,at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, or at least 90% of the enzymemixture. In some embodiments, the enzyme mixture comprises GH61, CBH2b,CBH1, Bgl, and at least one EG, in any suitable proportion for thedesired reaction.

In some embodiments, the enzyme mixture composition comprises isolatedcellulases in the following proportions by weight (wherein the totalweight of the cellulases is 100%), about 20%-10% of Bgl, about 30%-25%of CBH1a, about 10%-30% of GH61, about 20%-10% of EG, and about 20%-25%of CBH2b. In some embodiments, the enzyme mixture composition comprisesisolated cellulases in the following proportions by weight: about20%-10% of GH61, about 25%-15% of Bgl, about 20%-30% of CBH1a, about10%-15% of EG, and about 25%-30% of CBH2b. In some embodiments, theenzyme mixture composition comprises isolated cellulases in thefollowing proportions by weight: about 30%-20% of GH61, about 15%-10% ofBgl, about 25%-10% of CBH1a, about 25%-10% of CBH2b, about 15%-10% ofEG. In some embodiments, the enzyme mixture composition comprisesisolated cellulases in the following proportions by weight: about 40-30%of GH61, about 15%-10% of Bgl, about 20%-10% of CBH1a, about 20%-10% ofCBH2b, and about 15%-10% of EG. In some embodiments, the enzyme mixturecomposition comprises isolated cellulases in the following proportionsby weight: about 50-40% of GH61, about 15%-10% of Bgl, about 20%-10% ofCBH1a, about 15%-10% of CBH2b, and about 10%-5% of EG. In someembodiments, the enzyme mixture composition comprises isolatedcellulases in the following proportions by weight: about 10%-15% ofGH61, about 20%-25% of Bgl, about 30%-20% of CBH1a, about 15%-5% of EG,and about 25%-35% of CBH2b. In some embodiments, the enzyme mixturecomposition comprises isolated cellulases in the following proportionsby weight: about 15%-5% of GH61, about 15%-10% of Bgl, about 45%-30% ofCBH1a, about 25%-5% of EG, and about 40%-10% of CBH2b. In someembodiments, the enzyme mixture composition comprises isolatedcellulases in the following proportions by weight: about 10% of GH61,about 15% of Bgl, about 40% of CBH1a, about 25% of EG, and about 10% ofCBH2b.

In some embodiments, the enzyme component comprises more than one CBH1a,CBH2b, EG, Bgl, and/or GH61 enzyme (e.g., 2, 3, 4, or more differentvariants), in any suitable combination. In some embodiments, an enzymemixture composition of the invention further comprises at least oneadditional protein and/or enzyme. In some embodiments, enzyme mixturecompositions of the present invention further comprise at least oneadditional enzyme other than the GH61, Bgl, CBH1a, GH61, and/or CBH2b.In some embodiments, the enzyme mixture compositions of the inventionfurther comprise at least one additional cellulase, other than the GH61,Bgl, CBH1a, GH61, and/or CBH2b variant recited herein. In someembodiments, the GH61 polypeptide of the invention is also present inmixtures with non-cellulase enzymes that degrade cellulose,hemicellulose, pectin, and/or lignocellulose.

In some embodiments, GH61 polypeptide of the present invention is usedin combination with other optional ingredients such as at least onebuffer, surfactant, and/or scouring agent. In some embodiments, at leastone buffer is used with the GH61 polypeptide of the present invention(optionally combined with other enzymes) to maintain a desired pH withinthe solution in which the GH61 is employed. The exact concentration ofbuffer employed depends on several factors which the skilled artisan candetermine. Suitable buffers are well known in the art. In someembodiments, at least one surfactant is used in with the GH61 of thepresent invention. Suitable surfactants include any surfactantcompatible with the GH61 and, optionally, with any other enzymes beingused in the mixture. Exemplary surfactants include an anionic, anon-ionic, and ampholytic surfactants. Suitable anionic surfactantsinclude, but are not limited to, linear or branchedalkylbenzenesulfonates; alkyl or alkenyl ether sulfates having linear orbranched alkyl groups or alkenyl groups; alkyl or alkenyl sulfates;olefinsulfonates; alkanesulfonates, and the like. Suitable counter ionsfor anionic surfactants include, for example, alkali metal ions, such assodium and potassium; alkaline earth metal ions, such as calcium andmagnesium; ammonium ion; and alkanolamines having from 1 to 3 alkanolgroups of carbon number 2 or 3. Ampholytic surfactants suitable for usein the practice of the present invention include, for example,quaternary ammonium salt sulfonates, betaine-type ampholyticsurfactants, and the like. Suitable nonionic surfactants generallyinclude polyoxalkylene ethers, as well as higher fatty acidalkanolamides or alkylene oxide adduct thereof, fatty acid glycerinemonoesters, and the like. Mixtures of surfactants also find use in thepresent invention, as is known in the art.

In some embodiments, the cellulase enzyme mixtures of the presentinvention are produced in a fermentation process in which the fungalcell described herein above is grown in submerged liquid culturefermentation. It is intended that any suitable fermentation medium andprocess will find use in the present invention. In some embodiments,submerged liquid fermentations of fungal cells are conducted as a batch,fed-batch and/or continuous process. It is not intended that the presentinvention be limited to any particular fermentation medium, protocol,process, and/or equipment. In some embodiments, the fermentation mediumis a liquid comprising a carbon source, a nitrogen source, and othernutrients, vitamins and minerals which can be added to the fermentationmedia to improve growth and enzyme production of the host cell. In someembodiments, these other media components are added prior to,simultaneously with or after inoculation of the culture with the hostcell. In some embodiments, the carbon source comprises a carbohydratethat induces the expression of the cellulase enzymes from the fungalcell. For example, in some embodiments, the carbon source comprises oneor more of cellulose, cellobiose, sophorose, xylan, xylose, xylobioseand related oligo- or poly-saccharides known to induce expression ofcellulases and beta-glucosidase in such fungal cells. In someembodiments, the media comprise cellulose, while in some otherembodiments, the media do not comprise cellulose (i.e., measurableconcentrations of cellulose). In some further embodiments, the mediacomprise carbon sources such as glucose, dextrose, etc. However, it isnot intended that the present invention be limited to any specificcarbon and/or nitrogen source, as any suitable carbon and/or nitrogensource finds use in the present invention. Indeed, it is not intendedthat the present invention be limited to any particular medium, as anysuitable medium will find use in the desired setting.

In some embodiments utilizing batch fermentation, the carbon source isadded to the fermentation medium prior to or simultaneously withinoculation. In some other embodiments utilizing fed-batch and/orcontinuous operations, the carbon source is also supplied continuouslyor intermittently during the fermentation process. For example, in someembodiments, the carbon source is supplied at a carbon feed rate ofbetween about 0.2 and about 2.5 g carbon/L of culture/h, or any amounttherebetween. In some additional embodiments, the carbon source issupplied at a feed rate of between about 0.1 and about 10 g carbon/L ofculture/hour or at any suitable rate therebetween (e.g., about 0.1,about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, or about 10 g carbon/L of culture/h).

In some embodiments, the process for producing the enzyme mixture of thepresent invention is performed at a temperature from about 20° C. toabout 80° C., or any temperature therebetween, for example from about25° C. to about 65° C., or any temperature therebetween, or from about20° C., about 21° C., about 22° C., about 23° C., about 24° C., about25° C., about 26° C., about 27° C., about 28° C., about 29° C., about30° C., about 31° C., about 32° C., about 33° C., about 34° C., about35° C., about 36° C., about 37° C., about 38° C., about 39° C., about40° C., about 41° C., about 42° C., about 43° C., about 44° C., about45° C., about 46° C., about 47° C., about 48° C., about 49° C., about50° C., about 51° C., about 52° C., about 53° C., about 54° C., about55° C., about 56° C., about 57° C., about 58° C., about 59° C., about60° C., about 61° C., about 62° C., about 63° C., about 64° C., about65° C., about 66° C., about 67° C., about 68° C., about 69° C., about70° C., about 71° C., about 72° C., about 73° C., about 74° C., about75° C., about 76° C., about 77° C., about 78° C., about 79° C., about80° C., or any temperature therebetween.

In some embodiments, the methods for producing enzyme mixtures of thepresent invention are carried out at a pH from about 3.0 to about 8.0,or any pH therebetween, for example from about pH 3.5 to about pH 6.8,or any pH therebetween, for example from about pH 3.0, about 3.1, about3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8,about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1,about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4,6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1,about 7.2, about 7.3, about 7.4, 7.5, about 7.6, about 7.7, about 7.8,about 7.9, about 8.0, or any pH therebetween.

In some embodiments, the fermentation medium containing the fungal cellsare used following fermentation, while in some other embodiments, thefermentation medium containing the fungal cells and the enzyme mixtureare used, while in some additional embodiments, an enzyme mixture isseparated from the fungal cells (e.g., by filtration and/orcentrifugation), and the enzyme mixture in the fermentation medium isused, and in still additional embodiments, the fungal cells, enzyme(s),and/or enzyme mixtures are separated from the fermentation medium andthen used. Low molecular solutes such as unconsumed components of thefermentation medium may be removed by ultrafiltration or any othersuitable method. Any suitable method for separating cells, enzyme(s),and/or enzyme mixtures find use in the present invention. Indeed, it isnot intended that the present invention be limited to any particularpurification/separation method. In some additional embodiments, thefungal cells, enzyme(s) and/or enzyme mixtures are concentrated (e.g.,by evaporation, precipitation, sedimentation and/or filtration). In someembodiments, stabilizers are added to the compositions comprising fungalcells, enzyme(s), and/or enzyme mixtures. In some embodiments, chemicalssuch as glycerol, sucrose, sorbitol and the like find use to stabilizethe enzyme mixtures. In some additional embodiments, other chemicals(e.g., sodium benzoate and/or potassium sorbate), are added to theenzyme mixture to prevent growth of microbial contamination. In someadditional embodiments, additional components are present in thecompositions provided herein. It is not intended that the presentinvention be limited to any particular chemical and/or other components,as various components will find use in different settings. Indeed, it iscontemplated that any suitable component will find use in thecompositions of the present invention.

Methods for Generating Fermentable Sugars

The present invention provides methods for generating fermentablesugars, including but not limited to glucose. In some embodiments, themethods for generating glucose comprise contacting cellulose with fungalcells producing at least one enzyme, at least one enzyme, and/or atleast one enzyme mixture described herein. For example, in someembodiments, the process comprises contacting cellulose with an enzymemixture comprising two or more cellulose hydrolyzing enzymes, wherein atleast one of the two or more cellulose hydrolyzing enzymes is producedby a fungal cell as described herein.

In some embodiments, the method for generating fermentable sugars suchas glucose from cellulose using the enzyme mixture is batch hydrolysis,fed-batch hydrolysis, continuous hydrolysis, and/or a combinationthereof. In some embodiments, the hydrolysis reaction is agitated,stirred, unmixed, and/or a combination thereof.

The methods for generating fermentable sugars such as glucose fromcellulose are carried out at any suitable temperature known in the art.In some embodiments, a temperature of about 30° C. to about 80° C., orany temperature therebetween (e.g., a temperature of about 30° C., about31° C., about 32° C., about 33° C., about 34° C., about 35° C., about36° C., about 37° C., about 38° C., about 39° C., about 40° C., about41° C., about 42° C., about 43° C., about 44° C., about 45° C., about46° C., about 47° C., about 48° C., about 49° C., about 50° C., about51° C., about 52° C., about 53° C., about 54° C., about 55° C., about56° C., about 57° C., about 58° C., about 59° C., about 60° C., about61° C., about 62° C., about 63° C., about 64° C., about 65° C., about66° C., about 67° C., about 68° C., about 69° C., about 70° C., about71° C., about 72° C., about 73° C., about 74° C., about 75° C., about76° C., about 77° C., about 78° C., about 79° C., about 80° C., or anytemperature therebetween) find use.

In addition, any suitable pH finds use in the present invention. In someembodiments, a pH of about 3.0 to about 8.0, or any pH therebetween(e.g., about pH 3.5 to about pH 6.8, or any pH therebetween, for examplefrom about pH 3.0, about 31, about 3.2, about 3.3, about 3.4, about 3.5,about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8,about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1,about 6.2, about 6.3, about 6.4, 6.5, about 6.6, about 6.7, about 6.8,about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, 7.5,about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, or any pHtherebetween) finds use in the present invention,

In some embodiments, the initial concentration of cellulose in thehydrolysis reactor, prior to the start of hydrolysis, is about 0% (w/w),to about 0.1% (w/w), to about 15% (w/w), or any amount therebetween, forexample about 1%, about 2%, about 3%, about 4%, about 5%, about 6%,about 7%, about 8%, about 10%, about 11%, about 12%, about 13%, about14%, or about 15% or any amount therebetween. However, it is notintended that the present invention be limited to any particulartemperature, pH, time, nor cellulose concentration in the hydrolysisreaction, as any suitable temperature, pH, time, celluloseconcentration, as well as other parameters find use in the presentinvention.

In some embodiments, the dosage of the cellulase enzyme and/or cellulaseenzyme mixture used in the hydrolysis reaction is from about 0.1 toabout 100 mg protein per gram cellulose, or any suitable amounttherebetween (e.g., about 0.1, about 0.2, about 0.3, about 0.4, about0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, about 20, about 21, about 22, about 23, about 24, about25, about 26, about 27, about 28, about 29, about 30, about 31, about32, about 33, about 34, about 35, about 36, about 37, about 38, about39, about 40, about 41, about 42, about 43, about 44, about 45, about46, about 47, about 48, about 49, about 50, about 51, about 52, about53, about 54, about 55, about 56, about 57, about 58, about 59, 60,about 61, about 62, about 63, about 64, about 65, about 66, about 67,about 68, about 69, about 70, about 71, about 72, about 73, about 74,about 75, about 76, about 77, about 78, about 79, about 80, about 81,about 82, about 83, about 84, about 85, about 86, about 87, about 88,about 89%, about 90, about 91, about 100 mg protein per gram cellulose,or any amount therebetween. The hydrolysis is carried out for anysuitable time period. In some embodiments, the hydrolysis is performedfrom about 0.5 hours to about 300 hours, from about 15 hours to 100hours, or any time therebetween. In some embodiments, the hydrolysisreaction is performed for about 0.5, about 1, about 2, 3, about 4, about5, about 6, about 7, about 8, about 9, about 10, about 11, about 12,about 13, about 14, about 15, about 16, about 17, about 18, about 19,about 20, about 25, about 30, about 35, about 40, about 45, about 50,about 55, about 60, about 65, about 70, about 75, about 80, about 85,about 90, about 95, about 100, about 105, about 110, about 115, about120, about 125, about 130, about 135, about 140, about 145, about 150,about 155, about 160, about 165, about 170, about 175, about 180, about185, about 190, about 195, about 200, about 205, about 210, about 215,about 220, about 225, about 230, about 235, about 240, about 245, about250, about 255, about 260, about 265, about 270, about 275, about 280,about 285, about 290, about 295, about 300 hours, or any timetherebetween. It should be appreciated that the reaction conditions arenot meant to limit the invention in any manner and may be adjusted asdesired by those of skill in the art. Indeed, it is not intended thatthe present invention be limited to any particular hydrolysis reactiontime, protein concentration, or any other specific reaction parameter,as various reaction parameters and components find use in the presentinvention.

In some embodiments, the enzymatic hydrolysis is carried out in ahydrolysis reactor. In some embodiments, the enzyme and/or enzymemixture is added to the pretreated lignocellulosic feedstock (alsoreferred to as the “substrate”) prior to, during, or after the additionof the substrate to the hydrolysis reactor.

In some embodiments, various environmental conditions are adjustedaccording to any variety of methods known in the art in order tomaximize the formation of a hydrolysis product such as glucose. Forexample, temperature, pH, % dissolved oxygen, stirring speed can each beindependently adjusted. In some embodiments, the enzyme mixture is acell-free mixture, as described herein.

In some embodiments, the methods for generating glucose utilizingenzymes and/or enzyme mixtures comprising reduced glucose oxidase and/orreduced cellobiose dehydrogenase activity, as described herein, providehigher yields of glucose from the enzymatically hydrolyzed cellulosethan a corresponding method using an enzyme mixture with its fullcomplement of glucose oxidase or cellobiose dehydrogenase activity.Further, some embodiments of the methods provided herein result indecreased conversion of the cellobiose and glucose products in theenzymatic hydrolyzate to oxidized products such as gluconolactone,gluconate, gluconic acid cellobionolactone, and/or cellobionic acid.

In some embodiments of the methods provided herein utilizing thegenetically modified fungal cell(s), enzyme(s), and/or enzyme mixture(s)provided herein, improved glucose yield is measured and/or quantified.As described herein, glucose yield can be described in terms of theamount of generated glucose per theoretical maximum glucose yield, or interms of Gmax.

For example, as described in U.S. Pat. Nos. 6,090,595 and 7,419,809, thecellulose content can be determined by acid hydrolysis of the cellulose,followed by determination of glucose concentration, taking into accountthe water necessary to hydrolyze the cellulose. In one specific example,a slurry of feedstock is centrifuged, washed with water, and suspendedin sulfuric acid at a net sulfuric acid concentration of 70%. The slurryis incubated at 40° C. for 30 minutes, followed by diluting in deionizedwater to 2% sulfuric acid. At this time point, the samples aresteam-autoclaved at 121° C. for 1 hour, to convert the oligomers tomonomeric glucose. The glucose concentration is measured by HPLC orenzymatic assay as described below.

Alternatively, cellulose content can be analyzed by infraredspectroscopy as described in Example 1. For example, solids can bewashed and placed on the detection crystal of an infrared spectrometerand their absorbance measured between 500-4000 cm⁻¹.

Glucose levels can be quantified by any of a variety of methods known inthe art (See e.g., U.S. Pat. Nos. 6,090,595 and 7,419,809). For example,glucose concentrations can be determined using a coupled enzymatic assaybased on glucose oxidase and horseradish peroxidase (See e.g., Trinder,Ann. Clin. Biochem., 6:24-27 [1969]). Additional methods of glucosequantification include chromatographic methods, for example by HPLC (Seee.g., U.S. Pat. Nos. 6,090,595 and 7,419,809). Cellobiose levels can bemeasured by any number of HPLC methods known to those skilled in the art(See e.g., Kotiranta et al., Appl. Biochem. Biotechnol., 81: 81-90[1999].

Similarly, decreased conversion of cellobiose and glucose products tooxidized products such as cellobionolactone and gluconolactone can bequantified using any suitable method known in the art. For example,products of glucose or cellobiose oxidation can be detected andquantified using infrared spectroscopy, or by chromatographicmethodologies such as HPLC (See e.g., Rakotomanga et al., J. Chromatog.B. 4:277-284 [1991]; and Mansfield et al., App. Environ. Microbiol.,64:3804-3809 [1997]). Accordingly, total oxidation of glucose orcellobiose can be determined, for example, as a function of totaloxidation products per theoretical maximum glucose yield, or as afunction of Gmax.

The methods, fungal cells, enzymes, and enzyme mixtures provided hereininclude reduction or removal of glucose and/or cellobiose oxidizingenzyme activity from a cellulose hydrolyzing enzyme mixture, therebyimproving the yield of fermentable sugars such as glucose, xylose,and/or cellobiose during hydrolysis of cellulose. Advantageously, theprocesses and enzyme mixtures provided herein result in an increasedyield of glucose and/or cellobiose from the hydrolyzed cellulose and adecreased oxidation of the glucose and/or cellobiose to oxidized sugarproducts, such as gluconolactone, gluconate, gluconic acid,cellobionolactone, and/or cellobionic acid from the hydrolyzedcellulose, relative to an enzyme mixture with an unmodified amount ofglucose and/or cellobiose oxidizing enzyme activity, or relative to aparental enzyme mixture.

In some embodiments, the methods provided herein comprise contacting acellulose substrate with fungal cells producing at least one cellulosehydrolyzing enzyme, at least one cellulose hydrolyzing enzyme, and/or anenzyme mixture comprising two or more cellulose hydrolyzing enzymes. Insome embodiments, the enzyme mixtures are characterized in thatoxidation of cellobiose and/or glucose is reduced during hydrolysis ofcellulose, as described in greater detail herein, relative to an enzymemixture with an unmodified amount of glucose and/or cellobiose oxidizingenzyme activity, or relative to a parental enzyme mixture. In someembodiments, the processes provided herein comprise providing acellulose substrate, typically as an aqueous slurry, and providingatleast one enzyme mixture comprising at least two cellulose hydrolyzingenzymes, at least one cellulose hydrolyzing enzyme, and/or fungal cellsproducing at least one cellulose hydrolyzing enzyme. Thecellulose-containing slurry is introduced into a reaction vessel such asa hydrolysis reactor, and the at least one enzyme mixture comprising atleast two cellulose hydrolyzing enzymes, at least one cellulosehydrolyzing enzyme, and/or fungal cells producing at least one cellulosehydrolyzing enzyme is added to the vessel, in any order. After a periodduring which hydrolysis occurs, hydrolysis product in the form offermentable sugars such as glucose and/or cellobiose is produced, and,if desired, is recovered.

In some embodiments, the cellulosic substrate is provided in an aqueousslurry and added to a reaction vessel. The concentration of cellulosicfeedstock in the slurry depends on the material. In some embodiments,the concentration of cellulosic feedstock in the slurry is between about1% to about 30% (w/w) undissolved solids, or any concentrationtherebetween, for example, from about 5% to about 20%, or from about 10%to about 20% undissolved solids, or any amount therebetween. In someembodiments, the concentration of cellulosic feedstock in the slurrycomprises at least, at least about, up to, or about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, 13, 14, about 15, about 16, about 17, about 18, about 19,about 20, about 21, about 22, about 23, about 24, about 25, about 26,about 27, about 28, about 29, or about 30% undissolved solids (w/w).

Any suitable method known in the art for generating glucose fromcellulose using fungal cells producing at least onecellulose-hydrolyzing enzyme, at least one cellulose-hydrolyzing enzyme,and/or at least one enzyme mixtures find use in the present invention,including but not limited to batch hydrolysis, fed-batch hydrolysis, orcontinuous hydrolysis, as well as any suitable combination thereof. Insome embodiments of batch processes, all the necessary materials areplaced in a reactor at the start of the operation and the process isallowed to proceed until completion or until a desired endpoint, atwhich point the hydrolysis reaction is ended and in some embodiments,the product is harvested. In any batch process, one or more enzymes, thefungal cells producing at least one cellulose-hydrolyzing enzyme, and/orat least one enzyme mixture are added to the cellulose substrate before,during or after the introduction of the cellulose substrate to thereaction vessel. Thus, in some embodiments, the fungal cells, enzyme(s),and/or enzyme mixture(s) is added to the reaction vessel beforeintroducing cellulosic substrate to the reaction vessel. In some otherembodiments, the fungal cells, enzyme(s), and/or enzyme mixture(s) isadded to the reaction vessel simultaneously with cellulosic substrate.In some other embodiments, the fungal cells, enzyme(s) and/or enzymemixture(s) is added after introducing cellulosic substrate to thereaction vessel.

In some embodiments utilizing continuous process, cellulosic substrateis supplied and hydrolysis product is removed periodically orcontinuously at roughly volumetrically equal rates to maintain thehydrolysis reaction at a steady rate. Continuous processes can beperformed according to any of a variety of methods known in the art,including but not limited to upflow hydrolysis processes (See e.g., U.S.Pat. No. 7,727,746). However, it will be appreciated that any othersuitable continuous process finds use in the present invention.

In some embodiments, following their withdrawal from the reactor, atleast a portion of the unconverted solids are separated from the solublehydrolysis liquor. Removal of the unconverted solids is accomplishedusing any suitable method, including but not limited to solids-liquidseparation (e.g., by use of a filter press, belt filter, drum filter,vacuum filter, and/or membrane filter), centrifugation, settling (e.g.,by use of a settling tank or an inclined settler for example, asdisclosed in Knutsen and Davis, Appl., Biochem. Biotech.,98-100:1161-1172 [2002]; and Mores et al., Appl. Biochem. Biotech.,91-93:297-309 [2001]), clarification, or any other suitable process aswould be known in the art. Clarification may be carried out using anysuitable method known in the art. In some embodiments, a clarifiercomprising a number of inclined plates to facilitate the separation ofthe solids and liquid or other features that are known in the art ofsolids-liquid separation find use. The soluble glucose, essentially freeof undissolved solids, is then suitable for fermentation to ethanol. Theunconverted solids are primarily lignin, which can be further utilized.For example, the unconverted solids can be burned and used as fuel orconverted to generate electrical power.

In some embodiments, the unconverted solids comprise lignin, silicaand/or other solid material. It is not intended that the presentinvention be limited to any particular unconverted solid(s). As thecellulose in the feedstock is hydrolyzed and released from the solidparticles, the proportion of unconverted solids within thecellulose-containing solid particles increases. Depending on the densityand particle size, the unconverted solids may be removed with theproducts at or settle to the bottom of the reaction vessel in a sedimentor sludge. If a sludge layer forms at the bottom of the reactor due tovery heavy particles, any means known in the art may be employed toremove the sludge or sediment. For example, in some embodiments, ascraper is used to remove the sludge. In some alternative embodiments,the bottom of the reactor is tapered to provide a path in which theheaviest solids settle, and then be removed and sent for processing, asdesired.

In some embodiments, the fungal cells, enzyme(s), and/or enzymemixture(s) are recovered and reused after the hydrolysis is completed orduring the reaction. Recovery of the fungal cells, enzyme(s), and/orenzyme mixture(s) is accomplished using any suitable method known in theart. For example, in some embodiments, the fungal cells, enzyme(s),and/or enzyme mixture is removed from the hydrolysis liquor byprecipitation (e.g., pH precipitation, salt precipitation, and/ortemperature precipitation), extraction (e.g., solvent extraction),and/or filtration (e.g., ultrafiltration and/or microfiltration). It isnot intended that the present invention be limited to any particularrecovery method nor components. In some embodiments, the removed fungalcells, enzyme(s), and/or enzyme mixture(s) are added back to ahydrolysis reaction. In some embodiments utilizing ultrafiltration, themembrane has a molecular weight (MW) cut off of about 1,000, to about20,000. In some embodiments, the MW cut off is from about 5,000 to about10,000. In some embodiments, following recovery, the fungal cells,enzyme(s), and/or enzyme mixture(s) is recycled back into a reactor forfurther hydrolysis of additional feedstock. In some embodiments, therecycled enzyme(s) and/or enzyme mixture(s) are concentrated (e.g., byevaporation, precipitation, sedimentation and/or filtration). In someembodiments, chemicals such as glycerol, sucrose, sorbitol and the likeare added to stabilize the enzyme mixture. In some additionalembodiments, other chemicals, such as sodium benzoate or potassiumsorbate, are added to the enzyme(s) and/or enzyme mixture(s) to preventgrowth of microbial contamination.

In embodiments, the methods are conducted in a reaction volume within asuitable vessel. Any suitable vessel finds use in the present invention,including but not limited to flasks, bioprocess reactors, hydrolysisreactors, and the like. As used herein, the term “hydrolysis tower,”“hydrolysis reactor,” bioprocess reactor,” “hydrolysis tank,” and thelike refer to a reaction vessel of appropriate construction toaccommodate the hydrolysis of cellulosic slurry by at least onecellulase enzyme. It should be appreciated that one or more hydrolysisreactors may be utilized, such as one or more batch or continuousstirred reactors. In some embodiments, in which more than one hydrolysisreactor is employed, the reactors are run in a series of two or morethan two reactors, in which case the outlet of a first reactor feeds theinlet of a second reactor. Alternatively, in some embodiments, thereactors are run in parallel. Furthermore, in some embodiments, some ofthe reactors in the sequence are run in series, while others are run inparallel. Indeed, it is not intended that the present invention belimited to any particular reactor vessel, number of reactor vessels, norany configuration of multiple reactors.

In some embodiments, the cellulose hydrolysis reaction volume is egreater than about 0.01 mL, about 0.1 mL, about 1 mL, about 10 mL, about100 mL, about 1000 mL, or greater than about 5 L, about 10 L, about 25L, about 50 L, about 75 L, about 100 L, about L, about 200 L, about 250L, about 300 L, about 350 L, about 400 L, about 450 L, about 500 L,about 550 L, about 600 L, about 650 L, about 700 L, about 750 L, about800 L, about 850 L, about 900 L, about 950 L, about 1000 L, about 5000L, about 10,000 L, about 50,000 L, about 100,000 L, about 200,000 L,about 250,000 L, about 500,000 L, about 750,000 L, about 1,000,000 L, orgreater than about 1,000,000 L. Indeed, it is not intended that thepresent invention be limited to any particular reaction volume, as anysuitable/desired reaction volume funds use in the present invention. Insome embodiments, the hydrolysis reaction mixture is agitated, unmixed,or a combination thereof. For example, in some embodiments in which thehydrolysis reaction mixture is agitated, one or more impellers,agitators, eductors, and the like are used to mix the slurry. In someother embodiments, the hydrolysis reaction mixture is “unmixed,” in thatno mixing (i.e., no agitation) of the reactor contents takes placeduring the hydrolysis reaction. In some additional embodiments, otherfactors, such as the percentage of dissolved oxygen and/or stirringspeed are monitored and independently adjusted as needed.

Cellulosic Material

The cellulosic material used in the present invention can be anymaterial containing cellulose. The predominant polysaccharide in theprimary cell wall of biomass is cellulose, the second most abundant ishemicellulose, and the third is pectin. The secondary cell wall,produced after the cell has stopped growing, also containspolysaccharides and is strengthened by polymeric lignin covalentlycross-linked to hemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan, whilehemicelluloses include a variety of compounds, such as xylans,xyloglucans, arabinoxylans, and mannans in complex branched structureswith a spectrum of substituents. Although generally polymorphous,cellulose is found in plant tissue primarily as an insoluble crystallinematrix of parallel glucan chains. Hemicelluloses usually hydrogen bondto cellulose, as well as to other hemicelluloses, which help stabilizethe cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, herbaceous material,agricultural residue, forestry residue, municipal solid waste, wastepaper, and pulp and paper mill residue (See e.g., Wiselogel et al., inHandbook on Bioethanol, (Wyman, ed.), pp. 105-118, Taylor & Francis,Washington D.C. [1995]; Wyman, Biores. Technol., 50: 3-16 [1994]; Lynd,Appl. Biochem. Biotechnol., 24/25: 695-719 [1990]; Mosier et al., inAdvances in Biochemical Engineering/Biotechnology, (Scheper, ed.), Vol.65, pp. 23-40, Springer-Verlag, New York [1999]). It is understoodherein that the cellulose used in the present invention may be in theform of lignocellulose, a plant cell wall material containing lignin,cellulose, and/or hemicellulose in a mixed matrix. In some embodiments,the cellulosic material is lignocellulose.

In some embodiments, the pretreated lignocellulose used in the methodsof the present invention is a material of plant origin that, prior topretreatment, contains at least about 10%, about 20%, about 30%, orabout 40% cellulose (dry weight). In some embodiments, thelignocellulose comprises about 20, about 21%, about 22%, about 23%,about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%,about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%,about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%,about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%,about 76%, about 77%, about 78%, about 79%, about 80%, about 85, about90% cellulose (dry weight) or any percent therebetween, and at leastabout 10% lignin (dry wt) to about at least 12% (dry weight). However,it is not intended that the present invention be limited tolignocellulosic material comprising any particular percentage ofcellulose and/or lignin. In some embodiments, the lignocellulose issubjected to physical and/or chemical processes to make the fiber moreaccessible and/or receptive to the actions of cellulolytic enzymes. Insome embodiments, the lignocellulosic feedstock contains higher levelsof cellulose following pre-treatment, while in other embodiments, thecellulose level is not altered during the pretreatment process. Forexample, if acid pretreatment is employed, the hemicellulose componentis hydrolyzed, which increases the relative level of cellulose. In thiscase, in some embodiments, the pretreated feedstock contains greaterthan about 20% cellulose and greater than about 12% lignin.

Lignocellulosic feedstocks that find use in the present the presentinvention include, but are not limited to, agricultural residues such ascorn stover, wheat straw, barley straw, rice straw, oat straw, canolastraw, sugarcane straw and soybean stover; fiber process residues suchas corn fiber, sugar beet pulp, pulp mill fines and rejects or sugarcane bagasse; forestry residues such as aspen wood, other hardwoods,softwood, and sawdust; or grasses such as switch grass, miscanthus, cordgrass, and reed canary grass. In some embodiments, the lignocellulosicfeedstock is first subjected to size reduction by any of a variety ofmethods including, but not limited to, milling, grinding, agitation,shredding, compression/expansion, or other types of mechanical action.Size reduction by mechanical action can be performed by any type ofequipment adapted for the purpose, for example, but not limited to, ahammer mill.

Glucose and Cellobiose Oxidation

Use of the compositions and methods provided herein result in hydrolysisreactions having decreased oxidation of cellobiose and/or glucose duringhydrolysis of cellulose, relative to an enzyme mixture with anunmodified amount of glucose and/or cellobiose oxidizing enzymeactivity, or relative to a parental enzyme mixture. Therefore, in someembodiments, the enzyme(s) and/or enzyme mixtures used herein arecharacterized in that oxidation of cellobiose and/or glucose is reducedor eliminated.

In some embodiments, in some methods and enzyme mixtures of the presentinvention, the enzyme mixture is characterized in that, when the enzymemixture is contacted with cellobiose and/or cellulose and/or glucose(e.g., a cellobiose and/or cellulose and/or glucose substrate) no morethan about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%,about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, orabout 20% (wt %) of the cellobiose and/or glucose is oxidized. Forexample, when the enzyme mixture is contacted with cellobiose or glucoseno more than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%,or about 20% (wt %) of the cellobiose and/or glucose is oxidized to formcellobionolactone, cellobionic acid, gluconolactone, gluconate orgluconic acid. For example, no more than about 1%, about 2%, about 3%,about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10,about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about17%, about 18%, about 19%, or about 20% (wt %) of cellobiose and/orglucose is oxidized after about 1, about 5, about 10, about 15, about20, about 25, about 30, about 35, about 40, about 45, about 50, about55, or about 60 minutes, or after about 1.5, about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 11, about12, about 14, about 15, about 16, about 71, about 18, about 19, about20, about 25, about 30, about 35, about 40, about 45, about 50, about55, about 60, about 65, about 70, about 75, about 80, about 85, about90, about 95, about 100, about 105, about 110, about 115, about 120,about 125, about 130, about 135, about 140, about 145, about 150, about155, about 160, about 165, about 170, about 175, about 180, about 185,about 190, about 195, about 200, about 205, about 210, about 215, about220, about 225, about 230 about 235, about 240, about 245, about 250,about 255, about 260, about 265, about 270, about 275, about 280, about285, about 290, about 295, or about 300 hours or more. In someembodiments of the methods, the enzyme mixture is contacted with acellulose and/or glucose substrate for a set period of time underreaction conditions at or about the optimal for enzymatic cellulosehydrolysis activity,

In some embodiments in which the cellulose hydrolysis reaction isperformed in batch mode, no more than about 1%, about 2%, about 3%,about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%,about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about17%, about 18%, about 19%, or about 20% (wt %) of the cellobiose and/orglucose resulting from the hydrolysis of the cellulose substrate isoxidized after the termination of the batch mode cellulose hydrolysisreaction. In some embodiments in which the cellulose hydrolysis reactionis performed in continuous mode, no more than about 1%, about 2%, about3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%,about 17%, about 18%, about 19%, or about 20% (wt %) of the cellobioseand/or glucose resulting from the hydrolysis of the cellulose substrateis oxidized at the time the cellulose hydrolysis reaction reaches steadystate or quasi-steady state. In some embodiments, the initiation of thereaction can be the initial about 1, about 5, about 10, about 15, about20, about 25, about 30, about 35, about 40, about 45, about 50, about55, or 60 minutes, or about 1.5, about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14, about 15, about 16, about 17, about 18, about 19, about20, about 25, about 30, about 35, about 40, about 45, about 50, about55, about 60, about 65, about 70, about 75, about 80, about 85, about90, about 95, or about 100 hours, after the cellulose substrate andcellulase enzymes are first admixed.

Decreased conversion of cellobiose and glucose products to oxidizedproducts such as cellobionolactone and gluconolactone can be quantifiedby any of a variety of suitable methods known in the art. For example,products of glucose or cellobiose oxidation can be detected andquantified using infrared spectroscopy and/or chromatographic methodssuch as those as described in the Examples (See also, Rakotomanga etal., J. Chromatog. B. 4:277-284 [1991]; and Mansfield et al., App.Environ. Microbiol., 64:3804-3809 [1997]). Thus, in some embodiments,total oxidation of glucose and/or cellobiose are determined, forexample, as a function of total oxidation products per theoreticalmaximum glucose yield, or as a function of Gmax, as described herein.

In some embodiments, the assessment of the glucose and/or cellobioseoxidizing activity in an enzyme mixture is carried out under similarconditions of pH and temperature to those employed for the process ofhydrolyzing cellulose as described above. For example, in someembodiments, the assessment of the glucose and/or cellobiose oxidizingactivity of the enzyme mixture is carried out at a pH of about 3.0 toabout 8.0, or at a pH of about 5.0 to 6.0; and at a temperature of about30° C. to about 80° C., or at a temperature of about 50° C. to about 60°C. The concentration of glucose and/or cellobiose in the assessment istypically in a range of glucose and/or cellobiose concentrations thatwould be expected to be generated during the process of hydrolyzingcellulose as described herein. For example, in some embodiments, theglucose and/or cellobiose concentrations are from about 1 g/L to about500 g/L, or from about 10 g/L to about 200 g/L, or from about 30 g/L toabout 100 g/L. In some embodiments, an enzyme mixture is mixed with asolution containing both about 50% w/w glucose and about 5% w/wcellobiose or a solution containing about 50% w/w glucose alone, atabout pH 5.0 and about 60° C. for about 24 hr, as set forth in theExamples, after which glucose and/or cellobiose oxidation products arequantified, for example by IR or by HPLC as described in the Examples.In some embodiments, an enzyme mixture is mixed with a solutioncontaining about 100 g/L glucose at about pH 5.0 and about 55° C. forabout 24 hr.

Additionally, when comparing the glucose and/or cellobiose oxidizingactivity in an enzyme mixture to a reference (e.g., parental) enzymemixture, the conditions of pH, temperature, and glucose/cellobioseconcentration will depend upon such properties as the pH and temperatureoptima and stability, as well as the substrate affinity, of theparticular glucose and/or cellobiose oxidizing enzymes that are presentin the reference mixture and removed or inactivated in the enzymemixture of interest. In some embodiments, the comparison is carried outat a pH and temperature range that is optimal for the reference enzymemixture. In some other embodiments, the comparison is carried out atabout pH and within temperature range that is optimal for cellulosehydrolysis reaction of the modified enzyme mixture. In some embodiments,the assessment of the glucose and/or cellobiose oxidizing activity ofthe enzyme mixture is carried out at a pH of about 3.0 to about 8.0, orat a pH of about 5.0 to about 6.0; and at a temperature of about 30° C.to about 80° C., or about 50° C. to about 60° C. Further, it will beappreciated that the concentration of cellobiose and/or glucosesubstrate in such a comparison will generally be within a range so as toreadily detect oxidation products of the cellobiose and/or glucosesubstrate using the reference enzyme mixture. For example, in someembodiments, the concentration of cellobiose and/or glucose substrate isbelow a concentration that would cause substrate inhibition of theglucose and/or cellobiose oxidizing enzymes in the reference enzymemixture. Thus, in some embodiments, the glucose and/or cellobioseconcentrations generally range from about 1 g/L to about 300 g/L, orfrom about 10 g/L to about 100 g/L, or from about 30 g/L to about 70g/L. In some embodiments, A analysis of glucose and/or cellobioseoxidizing activity in an enzyme mixture is carried out under similarconditions, including, pH, temperature and glucose and/or cellobioseconcentrations, to those employed for the process of hydrolyzingcellulose. Further, it will be appreciated that identical conditionsshould be used to analyze the glucose and/or cellobiose oxidizingactivity of both modified enzyme mixture and of a reference enzymemixture.

In some embodiments, conversion of cellobiose and glucose products tooxidized products such as cellobionolactone and gluconolactone isindirectly quantified (e.g., by, measuring the total amount of glucoseand cellobiose produced relative to the amount of cellulose consumed).In some cellulose hydrolysis reactions, the only significant by-productsof the cellulose degradation reaction are oxidized products ofcellobiose or glucose, or transglycosylation products. The presence oftransglycosylation products can be distinguished from the presence ofoxidized products of cellobiose or glucose using a variety of methodsknown in the art or otherwise provided in the Examples herein. Forexample in some embodiments, only transglycosylation products, but notoxidized products, are acid-hydrolyzed to form only glucose. Thus, insome embodiments, the difference between the amount of celluloseconsumed and the amount of cellobiose and/or glucose present isdetermined, and reflects the amount of oxidized products of cellobioseor glucose, or reflects the amount of oxidized products of cellobiose orglucose and the amount of transglycosylation products. Methods ofquantifying cellulose and glucose are known in the art or are otherwiseprovided elsewhere herein.

Cellulose Conversion to Cellobiose and/or Glucose

In some embodiments, the methods for generating glucose, as describedherein, using the enzyme mixture with reduced or removed glucose and/orcellobiose oxidizing enzyme activity, and the enzyme mixture itself, arecharacterized as providing a higher yield of cellobiose and/or glucosefrom the enzymatically hydrolyzed cellulose than a corresponding processusing an enzyme mixture, or an enzyme mixture itself, with an unmodifiedamount of glucose and/or cellobiose oxidizing enzyme activity. In someembodiments, the methods for generating glucose provided herein usingthe enzyme mixture with reduced or removed glucose and/or cellobioseoxidizing enzyme activity, and the enzyme mixture itself, arecharacterized in providing a higher yield of cellobiose and/or glucosefrom the enzymatically hydrolyzed cellulose than a corresponding processusing a reference enzyme mixture under essentially the same pH,temperature and other conditions including, but not limited to,feedstock concentration, mineral concentration, stir rate, percentoxygenation, and other conditions relevant to those skilled in the artfor reproducing cellulose hydrolysis reactions.

In reference to an “unmodified amount of glucose and/or cellobioseoxidizing enzyme activity,” this phrase refers to an enzyme mixtureobtained from a biological source organism in which the biologicalsource organism has not been genetically modified or otherwise modifiedin such a way as to specifically target and thereby reduce thesecretion, expression or activity of a glucose and/or cellobioseoxidizing enzyme, and/or the enzyme mixture has not been manipulated insuch a way as to thereby reduce the amount or activity of a glucoseand/or cellobiose oxidizing enzyme. In reference to a reference enzymemixture, this term refers to an enzyme mixture obtained from a referencebiological source organism that is the same biological source organismas the biological source organism that provides the enzyme mixture withreduced or removed glucose and/or cellobiose oxidizing enzyme activity,where the biological source organism has not been genetically modifiedin such a way as to specifically target and thereby reduce thesecretion, expression or activity of a glucose and/or cellobioseoxidizing enzyme, and the enzyme mixture has not been manipulated insuch a way as to thereby reduce the amount or activity of a glucoseand/or cellobiose oxidizing enzyme.

As used herein in reference to a percentage of cellulose hydrolyzed bythe enzyme mixture present in the form of cellobiose and/or glucose,these percentages reflect a weight percent based on the dry weight ofthe hydrolyzed cellulose.

Thus, in some embodiments of the methods provided herein, when thefungal cells producing at least one enzyme, at least one enzyme, and/orat least one enzyme mixture is contacted with cellulose, at least about80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or about 100% (wt %) of the cellulose hydrolyzed by the enzyme mixtureis present in the form of cellobiose and/or glucose. In someembodiments, the fungal cells producing at least one enzyme, at leastone enzyme, and/or at least one enzyme mixture is contacted withcellulose for a period of time of at least about 1, about 5 about 10,about 15, about 20, about 25, about 30, about 35, about 40, about 45,about 50, about 55, or about 60 minutes, or at least about 1.5, about 2,about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10,about 11, about 12, about 13, about 14, about 15, about 16, about 17,about 18, about 19, about 20, about 25, about 30, about 35, about 40,about 45, about 50, about 55, about 60, about 65, about 70, about 75,about 80, about 85, about 90, about 95, about 100, about 105, about 110,about 115, about 120, about 125, about 130, about 135, about 140, about145, about 150, about 155, about 160, about 165, about 170, about 175,about 180, about 185, about 190, about 195, about 200, about 205, about210, about 215, about 220, about 225, about 230, about 235, about 240,about 245, about 250, about 255, about 260, about 265, about 270, about275, about 280, about 285, about 290, about 295, or about 300 hours ormore.

In some embodiments in which the cellulose hydrolysis reaction isperformed in batch mode, at least about 80%, about 81%, about 82%, about83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, 9 about 8%, about 99%, or about 100% (wt %) of thecellulose hydrolyzed by the enzyme mixture is present in the form ofcellobiose and/or glucose after the termination of the batch modecellulose hydrolysis reaction. In some embodiments in which thecellulose hydrolysis reaction is performed in continuous mode, at leastabout 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about99% or about 100% (wt %) of the cellulose hydrolyzed by the enzymemixture is present in the form of cellobiose and/or glucose at the timethe cellulose hydrolysis reaction reaches steady state or quasi-steadystate. In some embodiments, the initiation of the reaction can be theinitial about 1, about 5, about 10, about 15, about 20, about 25, about30, about 35, about 40, about 45, about 50, about 55, or about 60minutes, or about 1.5, about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, about 10, about 12, about 13, about 14, about15, about 16, about 17, about 18, about 19, about 20, about 25, about30, about 35, about 40, about 45, about 50, about 55, about 60, about65, about 70, about 75, about 80, about 85, about 90, about 95, or about100 hours, after the cellulose substrate and cellulase enzymes are firstadmixed.

Enzyme Mixtures with Reduced Glucose and/or Cellobiose Oxidizing EnzymeActivity

The present invention provides enzyme mixtures with reduced glucoseand/or cellobiose oxidizing enzyme activity, which, when contacted withcellulose, result in a higher yield of glucose from the hydrolysis ofcellulose than a corresponding method using an enzyme mixture with anunmodified amount of glucose and/or cellobiose oxidizing enzymeactivity. In some embodiments, the enzyme mixture is characterized asproviding a higher yield of cellobiose and/or glucose from theenzymatically hydrolyzed cellulose than a corresponding process using areference enzyme mixture. In some embodiments, the enzyme mixture ischaracterized as causing decreased conversion of the cellobiose andglucose products in the enzymatic hydrolysate to oxidized products suchas cellobionolactone and gluconolactone relative to an enzyme mixturewith an unmodified amount of glucose and/or cellobiose oxidizing enzymeactivity, or relative to a reference enzyme mixture.

In some embodiments, an enzyme mixture with reduced glucose and/orcellobiose oxidizing enzyme activity is treated to reduce the amount ofglucose and/or cellobiose oxidizing enzyme in the enzyme mixture. Itwill be readily appreciated that any of a variety of technologies knownin the art can be employed to reduce the amount of glucose and/orcellobiose oxidizing enzyme from the enzyme mixture, including but notlimited to purification processes that selectively remove one or moreglucose and/or cellobiose oxidizing enzyme activity from the enzymemixture. In some embodiments, inhibitors of glucose and/or cellobioseoxidizing enzyme activity are added to the enzyme mixture. Additionalembodiments include the use of genetically modified fungal cells toreduce the amount of one or more endogenous glucose and/or cellobioseoxidizing enzymes secreted by the fungal cell.

Purification Processes. In some embodiments, the enzyme mixture issubjected to a purification process to selectively separate one or moreglucose and/or cellobiose oxidizing enzymes from the enzyme mixture. Insome embodiments, the purification process comprises removing theglucose and/or cellobiose oxidizing enzyme from the enzyme mixture usingan affinity-based stationary phase. Affinity-based purificationtechnologies are well known in the art, and include any method toselectively bind a component of a biological mixture to a solid supportbased on a highly specific biological interaction such as that betweenantigen and antibody or enzyme and substrate. Thus, affinity-basedmethodologies include contacting the enzyme mixture with beads or anyother suitable solid support that comprises antibodies or othermolecules that selectively bind to and immobilize the glucose and/orcellobiose oxidizing enzyme, while the remaining components of theenzyme mixture remain in solution. Some examples include chromatographymethods either in batch form or column form. The solid support cancomprise individual particles (e.g., chromatography resin beads) orcontiguous supports (e.g., arrays). Ligands immobilized on a solidsupport matrix can then be employed to purify targets from complexsolutions. Conventional chromatography supports, as well as standardmethods for grafting antibodies well known in the art and are describedin numerous standard textbooks. These methods include the use of tubes,particles such as beads and any other suitable solid support. Forexample, particles are available in a large variety of differentmaterials, including silica, glass, cellulose, agarose, and a widevariety of different polymers, including polystyrenepolymethylmethacrylate, polyacrylamide, agarose, hydrogel, acrylicresins, and other types of gels used for electrophoresis. These supportsmay be purchased with ligands pre-attached or alternatively, the ligandscan be indirectly attached or directly immobilized on the support usingstandard methods well known to those skilled in the art (See e.g.,Biancala et al., Lett. Peptide Sci., 7:297 [2000]; MacBeath et al.,Science, 289:1760-1763 [2000]; Cass et al., (eds.), Proc. Thirteenth Am.Peptide Symp., Leiden, Escom, 975-979 [1994]; U.S. Pat. No. 5,576,220;Cook et al, Tetrahed. Lett., 35:6777-6780 [1994]).

In some embodiments, the stationary phase comprises antibodies and/orany other molecule that selectively binds the glucose and/or cellobioseoxidizing enzyme, such an antibody fragments (e.g., a Fab, Fab′ orF(ab′)₂). Strategies for depletion of specific proteins in a complexmixture of proteins are well known (See e.g., Bjorhall et al. Proteomics5:307-317 [2005]). In some embodiments, the stationary phase comprisesantibodies directed toward glucose oxidase (EC 1.1.3.4), cellobiosedehydrogenase (EC 1.1.99.18), pyranose oxidase (EC1.1.3.10),glucooligosaccharide oxidase (EC 1.1.99.B3), pyranose dehydrogenase (EC1.1.99.29) or glucose dehydrogenase (EC 1.1.99.10).

In some embodiments, the stationary phase comprises molecules thatselectively bind the glucose and/or cellobiose oxidizing enzyme(s). Forexample, in some embodiments, a binding protein, substrate, substrateanalogue or other small molecule is coupled to the stationary phase toselectively bind the enzyme of interest. In some embodiments, thestationary phase comprises a glucose and/or cellobiose linked to thestationary phase. In some other embodiments, the stationary phasecomprises a flavin adenine dinucleotide (FAD) linked to the stationaryphase.

It will be appreciated that any of a variety of other purificationmethodologies are useful in the selective removal of one or more glucoseand/or cellobiose oxidizing enzymes from the enzyme mixture. Forexample, in some embodiments, the purification methodology comprisesfractionation methods including selective precipitation such as ammoniumsulfate precipitation, isoelectric precipitation, selective thermaldenaturation, or any other method which selectively precipitates glucoseand/or cellobiose oxidizing enzymes from the enzyme mixture, whileleaving other components of the enzyme mixture in solution, or viceversa. In some other embodiments, the purification methodologiescomprise chromatographic methods including gel filtration, sizeexclusion, anionic exchange, cationic exchange, gel electrophoresis,and/or other chromatic separation method known in the art for physicallyseparating proteins.

Oxidase Inhibitors. In some embodiments, reducing the amount of glucoseand/or cellobiose oxidizing enzyme activity from the enzyme mixtureemploys the addition of one or more glucose and/or cellobiose oxidizingenzyme inhibitor(s) to the enzyme mixture Inhibitors of glucose and/orcellobiose oxidizing enzymes range from broad-spectrum oxidaseinhibitors to specific inhibitors of glucose and/or cellobiose oxidizingenzymes, as described herein.

In some embodiments, a broad-spectrum oxidase inhibitor is added to theenzyme mixture. Broad-spectrum oxidase inhibitors are well-known in theart. Some examples include but are not limited to mercuric chloride,silver sulphate, hydrazine compounds such as aminoguanidine,semicarbazide, benserazide, oxalic dihydrazide, hydralazine,phenylhydrazine, carbidopa, diaminoguanidine, and copper chelators suchas desferrioxamine, EDTA, sodium azide, potassium cyanide, triene 5,o-phenanthroline, histidine and a number of metal anions such as Ag⁺,Hg²⁺, and Zn²⁺.

In some embodiments, a specific inhibitor of glucose and/or cellobioseoxidizing enzymes is added to the enzyme mixture. Specific inhibitors ofcellobiose dehydrogenase include but are not limited to substrateanalogues and other specific inhibitors such as cellobioimidazole,gentiobiose, lactobiono-1,5-lactone, celliobono-1,5-lactone,tri-N-acetylchitortriose, methyl-beta-D-cellobiosidase, 2,2-bipyridine,and/or cytochrome C. Specific inhibitors of glucose oxidase, pyranoseoxidase, glucooligosaccharide oxidase, pyranose dehydrogenase, andglucose dehydrogenase include but are not limited to substrate analoguesand other specific inhibitors.

Genetically Modified Fungal Cells. In some embodiments, the enzymemixture is produced by a fungal cell that has been genetically modifiedto reduce the amount of the endogenous glucose and/or cellobioseoxidizing enzyme activity that is secreted by the fungal cell.

Genetic modifications contemplated herein reduce the enzymatic activityof glucose and/or cellobiose oxidizing enzymes produced by a fungalcell. Any glucose and/or cellobiose oxidizing enzyme known in the artcan be targeted for reduction of activity by genetic modification. Forexample, glucose and/or cellobiose oxidizing enzymes include glucoseoxidase (EC 1.1.3.4), cellobiose dehydrogenase (EC 1.1.99.18), pyranoseoxidase (EC 1.1.3.10), glucooligosaccharide oxidase (EC 1.1.99.B3),pyranose dehydrogenase (EC 1.1.99.29), and glucose dehydrogenase (EC1.1.99.10). Each of these glucose and/or cellobiose oxidizing enzymesare described in the four Provisional applications to which the presentapplication claims priority (e.g., U.S. Prov. Patent Appln. Ser. Nos.61/409,186, 61/409,217, 61/409,472, and 61/409,480, all of which werefiled on Nov. 2, 2010 and are hereby incorporated by reference herein).

Pretreatment.

In some embodiments, a substrate of the enzyme mixture comprisespretreated cellulosic material. Thus, for example, in processesdescribed herein, any pretreatment process known in the art can be usedto disrupt plant cell wall components of cellulosic material (See e.g.,Chandra et al., Adv. Biochem. Engin./Biotechnol., 108: 67-93 [2007];Galbe and Zacchi, Adv. Biochem. Engin./Biotechnol., 108: 41-65 [2007];Hendriks and Zeeman, Biores. Technol., 100:10-18 [2009]; Mosier et al.,Biores. Technol., 96: 673-686 [2005]; Taherzadeh and Karimi, Int. J.Mol. Sci., 9:1621-1651 [2008]; and Yang and Wyman, Biofuels Bioprod.Bioref.-Biofpr. 2: 26-40 [2008]).

In some embodiments, the cellulosic material is subjected to particlesize reduction, pre-soaking, wetting, washing, and/or conditioning priorto pretreatment, using any of a variety of methods known in the art.

In some embodiments, conventional pretreatments that find use in thepresent invention include, but are not limited to, steam pretreatment(with or without explosion), dilute acid pretreatment, hot waterpretreatment, alkaline pretreatment, lime pretreatment, wet oxidation,wet explosion, ammonia fiber expansion, dilute ammonia pretreatment,organosolv pretreatment, and/or biological pretreatment. Additionalpretreatments include, but are not limited to ammonia percolation,ultrasound, electroporation, microwave, supercritical CO₂, supercriticalH₂O, ozone, and gamma irradiation pretreatments.

In some embodiments, the cellulosic material is pretreated beforehydrolysis and/or fermentation. In some embodiments, pretreatment isperformed prior to the hydrolysis. In some alternative embodiments, thepretreatment is carried out simultaneously with enzyme hydrolysis torelease fermentable sugars, such as glucose, xylose, and/or cellobiose.In some embodiments, the pretreatment step itself results in someconversion of biomass to fermentable sugars (even in absence ofenzymes).

Steam Pretreatment. In steam pretreatment, cellulosic material is heatedto disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions(e.g., hemicelluloses), accessible to enzymes. Cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably done at about 140° C. to about 230° C., or from about 160° C.to about 200° C., or about 170° C. to about 190° C., where the optimaltemperature range depends on any addition of a chemical catalyst. Insome embodiments, residence time for the steam pretreatment is fromabout 1 to about 15 minutes, or about 3 to about 12 minutes, or about 4to about 10 minutes, where the optimal residence time depends ontemperature range and any addition of a chemical catalyst. Steampretreatment allows for relatively high solids loadings, so thatcellulosic material is generally only moist during the pretreatment. Thesteam pretreatment is often combined with an explosive discharge of thematerial after the pretreatment, which is known as steam explosion, thatis, rapid flashing to atmospheric pressure and turbulent flow of thematerial to increase the accessible surface area by fragmentation (Seee.g., U.S. Pat. No. 4,451,648; Duff and Murray, Biores. Technol., 855:1-33 [1996]; Galbe and Zacchi, Appl. Microbiol. Biotechnol., 59:618-628[2002]; and U.S. Pat. Appln. Pub. No. 2002/0164730). During steampretreatment, hemicellulose acetyl groups are cleaved and the resultingacid autocatalyzes partial hydrolysis of the hemicellulose tomonosaccharides and oligosaccharides. Lignin is removed to only alimited extent.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 3% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (See e.g.,Ballesteros et al., Appl. Biochem. Biotechnol., 129-132: 496-508 [2006];Varga et al., Appl. Biochem. Biotechnol., 113-116:509-523 [2004];Sassner et al., Enzyme Microb. Technol., 39:756-762 [2006]).

Chemical Pretreatment. Examples of suitable chemical pretreatmentprocesses include, but are not limited to, dilute acid pretreatment,dilute alkali pretreatment (See e.g., U.S. Pat. Appln. Pub. Nos.2007/0031918 and 2007/0037259), lime pretreatment, wet oxidation,ammonia fiber/freeze explosion or expansion (AFEX), ammonia percolation(APR), and organosolv pretreatments.

In dilute acid pretreatment, cellulosic material is mixed with diluteacid, typically H₂SO₄, and water to form a slurry, heated by steam tothe desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs (e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors; Seee.g., Duff and Murray, Biores. Technol., 855: 1-33 [1996]; Schell etal., Biores. Technol., 91:179-188 [2004]; and Lee et al., Adv. Biochem.Eng. Biotechnol., 65: 93-115 [1999]).

Any suitable methods for pretreatment under alkaline conditions alsofind use. These alkaline pretreatments include, but are not limited to,lime pretreatment, wet oxidation, ammonia percolation (APR), ammoniafiber/freeze expansion (AFEX) and dilute ammonia pretreatment.

Lime pretreatment is performed with calcium carbonate, sodium hydroxide,or ammonia at low temperatures of 85-150° C. and residence times from 1hour to several days (See e.g., Wyman et al., Biores. Technol.,96:1959-1966 [2005]; Mosier et al., Biores. Technol., 96:673-686 [2005];WO 2006/110891; WO 2006/11899; WO 2006/11900; and WO 2006/110901).

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (See e.g., Schmidt and Thomsen,Biores. Technol., 64:139-151 [1998]; Palonen et al., Appl. Biochem.Biotechnol., 117:1-17 [2004]; Varga et al., Biotechnol. Bioeng.,88:567-574 [2004]; Martin et al., J. Chem. Technol. Biotechnol.,81:1669-1677 [2006]). In some embodiments, the pretreatment is performedat about 1% to about 40% dry matter, or about 2% to about 30% drymatter, or about 5% to about 20% dry matter, and often the initial pH isincreased by the addition of alkali such as sodium carbonate.

A modification of the wet oxidation pretreatment method, known as “wetexplosion” (i.e., the combination of wet oxidation and steam explosion),can handle dry matter up to 30%. In wet explosion, the oxidizing agentis introduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (Seee.g., WO 2006/032282).

Ammonia fiber expansion (AFEX) involves treating cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-100°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (See e.g., Gollapalli et al., Appl.Biochem. Biotechnol., 98:23-35 [2002]; Chundawat et al., Biotechnol.Bioeng., 96:219-231 [2007]; Alizadeh et al., Appl. Biochem. Biotechnol.,121:1133-1141 [2005]; Teymouri et al., Biores. Technol., 96:2014-2018[2005]). AFEX pretreatment results in the depolymerization of celluloseand partial hydrolysis of hemicellulose. Lignin-carbohydrate complexesare cleaved. Dilute ammonia pretreatment utilizes more dilute solutionsof ammonia than AFEX and may be conducted at a temperature of about100-150° C., or any temperature therebetween (See e.g., U.S. Pat. Appln.Pub. Nos. 2007/0031918 and 2007/0037259). The duration of the diluteammonia pretreatment may be 1-20 minutes, or any duration therebetween.

Organosolv pretreatment delignifies cellulosic material by extractionusing aqueous ethanol (40-60% ethanol) at 160-200° C. for 30-60 minutes(See e.g., Pan et al., Biotechnol. Bioeng., 90:473-481 [2005]; Pan etal., Biotechnol. Bioeng., 94:851-861 [2006]; and Kurabi et al., Appl.Biochem. Biotechnol., 121:219-230 [2005]). Sulphuric acid is usuallyadded as a catalyst. In organosolv pretreatment, the majority ofhemicellulose is removed.

There are various other suitable methods for pretreatment that find usein the present invention (See e.g., Schell et al., Appl. Biochem.Biotechnol., 105-108:69-85 [2003]; and Mosier et al., Biores. Technol.,96:673-686 [2005]; and U.S. Pat. Appln. Publ. No. 2002/0164730).

In some embodiments, the chemical pretreatment is carried out as an acidtreatment. In some alternative embodiments, it is a continuous diluteand/or mild acid treatment. In some embodiments, the acid is sulfuricacid, but other acids also find use, including but not limited tonitricacid, phosphoric acid, hydrogen chloride, and/or mixtures thereof. Mildacid treatment is conducted in the pH range of about 1 to about 5, orabout 1 to about 4, or about 1 to about 3. In some embodiments, the acidconcentration is in the range from about 0.01 to about 20 wt % acid, orabout 0.05 to about 10 wt % acid, or about 0.1 to about 5 wt % acid, orabout 0.2 to about 2.0 wt % acid. The acid is contacted with cellulosicmaterial and held at a temperature in the range of about 160° C. toabout 220° C., or for about 165° C. to about 195° C., for periodsranging from seconds to minutes (e.g., about 1 second to about 60minutes).

In some other embodiments, pretreatment is carried out as an ammoniafiber expansion step (AFEX pretreatment step).

In some embodiments, pretreatment takes place in an aqueous slurry. Insome embodiments, cellulosic material is present during pretreatment inamounts between about 10 to about 80 wt %, or about 20 to about 70 wt %,or between about 30 to about 60 wt %, such as around 50 wt %. In someembodiments, the pretreated cellulosic material is unwashed, while insome other embodiments, it is washed using any method known in the art(e.g., washed with water).

Mechanical Pretreatment. Any suitable methods of mechanical pretreatmentfind use in the present invention.

Physical Pretreatment. As used herein, the term “physical pretreatment”refers to any pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from cellulosic material. Forexample, in some embodiments, physical pretreatment involves irradiation(e.g., microwave irradiation), steaming/steam explosion,hydrothermolysis, and combinations thereof.

In some embodiments, physical pretreatment involves high pressure and/orhigh temperature (steam explosion). In some embodiments, “high pressure”means pressure in the range of about 300 to about 600 psi, or about 350to about 550 psi, or about 400 to about 500 psi, such as around 450 psi.In some other embodiments, “high temperature” means temperatures in therange of about 100° C. to about 300° C., or about 140° C. to about 235°C. In some embodiments, mechanical pretreatment is performed in abatch-process, steam gun hydrolyzer system that uses high pressure andhigh temperature as defined above (e.g., a Sunds Hydrolyzer availablefrom Sunds Defibrator AB, Sweden).

Combined Physical and Chemical Pretreatment. In some embodiments,cellulosic material is pretreated both physically and chemically. Forinstance, in some embodiments, the pretreatment step involves dilute ormild acid treatment and high temperature and/or pressure treatment. Thephysical and chemical pretreatments can be carried out sequentially orsimultaneously, as desired. In some embodiments, a mechanicalpretreatment is also included.

Accordingly, in some embodiments, cellulosic material is subjected tomechanical, chemical, and/or physical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment. In some embodiments, biological pretreatmentprocesses find use in the present invention. Biological pretreatmenttechniques can involve applying lignin-solubilizing microorganisms (Seee.g., Hsu, “Pretreatment of Biomass,” in Handbook on Bioethanol:Production and Utilization (Wyman, ed.), Taylor & Francis, Washington,D.C., pp. 179-212 [1996]; Ghosh and Singh, Adv. Appl. Microbiol.,39:295-333 [1993]; McMillan, “Pretreating Lignocellulosic Biomass: aReview, in Enzymatic Conversion of Biomass for Fuels Production (Himmelet al. eds.), ACS Symposium Series 566, American Chemical Society,Washington, D.C., chapter 15 [1994]; Gong et al., 65: 207-241 [1999];Olsson and Hahn-Hagerdal, Enz. Microb. Tech., 18:312-331 [1996]; andVallander and Eriksson, Adv. Biochem. Eng./Biotechnol., 42:63-95[1990]).

In some embodiments, the soluble compounds derived from pretreatmentprocess are subsequently separated from the solids. For example, in someembodiments, the separation step comprises one or more of standardmechanical means such as screening, sieving, centrifugation, and/orfiltration to achieve the separation. In some other embodiments, thesoluble compounds are not separated from the solids followingpretreatment. It will be appreciated that pretreatment may be conductedas a batch, fed-batch or continuous process. It will also be appreciatedthat pretreatment may be conducted at low, medium or high solidsconsistency (See e.g., WO 2010/022511).

Fermentation

In some embodiments, the methods for generating glucose provided hereinfurther comprise fermentation of the resultant fermentable sugars (e.g.,glucose) to an end product. Especially suitable fermenting organisms areable to ferment (i.e., convert), sugars, such as glucose, fructose,maltose, xylose, mannose and/or arabinose, directly or indirectly intoat least one desired end product.

In some embodiments, yeast that find use in the present inventioninclude, but are not limited to strains of the genus Saccharomyces(e.g., strains of Saccharomyces cerevisiae and Saccharomyces uvarum),strains of the genus Pichia (e.g., Pichia stipitis, such as Pichiastipitis CBS 5773 and Pichia pastoris), strains of the genus Candida(e.g., Candida utilis, Candida arabinofermentans, Candida diddensii,Candida sonorensis, Candida shehatae, Candida tropicalis, and Candidaboidinii). Other fermenting organisms include, but are not limited tostrains of Zymomonas, Hansenula (e.g., Hansenula polymorphs andHansenula anomala), Kluyveromyces (e.g., Kluyveromyces fragilis), andSchizosaccharomyces (e.g., Schizosaccharomyces pombe).

Suitable bacterial fermenting organisms include, but are not limited tostrains of Escherichia (e.g., Escherichia coli), strains of Zymomonas(e.g., Zymomonas mobilis), strains of Zymobacter (e.g., Zymobactorpalmae), strains of Klebsiella (e.g., Klebsiella oxytoca), strains ofLeuconostoc (e.g., Leuconostoc mesenteroides), strains of Clostridium(e.g., Clostridium butyricum), strains of Enterobacter (e.g.,Enterobacter aerogenes), and strains of Thermoanaerobacter (e.g.,Thermoanaerobacter BG1L1; See, Appl. Microbiol, Biotech. 77: 61-86;Thermoanaerobacter ethanolicus, Thermoanaerobacterthermosaccharolyticum, and Thermoanaerobacter mathranii). Strains ofLactobacillus also find use in the present invention, as well as strainsof Corynebacterium glutamicum R, Bacillus thermoglucosidaisus, andGeobacillus thermoglucosidasius. Indeed, it is not intended that thepresent invention be limited to any particular fermenting organism.

The fermentation conditions depend on the desired fermentation productand can easily be determined by one of ordinary skill in the art. Insome embodiments involving ethanol fermentation by yeast, thefermentation occurs for between about 1 and about 120 hours, or betweenabout 12 and about 96 hours. In some embodiments, the fermentation iscarried out at a temperature between about 20 to about 40° C., or about26 to about 34° C., or about 32° C. In some embodiments, thefermentation pH is from pH about 3 to about 7, or about pH about 4 toabout 6.

In some embodiments, enzymatic hydrolysis and fermentation are conductedin separate vessels so that each biological reaction can occur under itsrespective optimal conditions (e.g., temperature). In some otherembodiments, the process for producing glucose from cellulose describedherein is conducted simultaneously with fermentation in a simultaneoussaccharification and fermentation (SSF). SSF is typically carried out attemperatures of about 28° C. to about 50° C., or about 30° C. to about40° C., or about 35° C. to about 38° C., which is a compromise betweenthe about 50° C. optimum for most cellulase enzyme mixtures and theabout 28° C. to about 30° C. optimum for growth of most yeast.

Accordingly, in some embodiments, the methods for generating glucosefurther comprise fermentation of the glucose to at least one endproduct. It is not intended that the present invention be limited to anyparticular end product, as the methods of the present invention aresuitable to produce a variety of end products. In some embodiments, theend products include, but are not limited to fuel alcohols and/orprecursor industrial chemicals. For example, in some embodiments, thefermentation products include precursor industrial chemicals such asalcohols (e.g., ethanol, methanol, butanol); organic acids (e.g.,butyric acid, citric acid, acetic acid, itaconic acid, lactic acid,gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamicacid); gases (e.g., H₂ and CO₂); antibiotics (e.g., penicillin andtetracycline); enzymes; vitamins (e.g., riboflavin, 812, beta-carotene);and/or hormones. In some embodiments, the end product is a fuel alcohol.Suitable fuel alcohols are known in the art and include, but are notlimited to lower alcohols such as methanol, ethanol, butanol, and propylalcohols.

Increased Expression of Saccharide Hydrolysis Enzymes

In some embodiments provided herein, the fungal cell is furthergenetically modified to increase its production of one or moresaccharide hydrolysis enzymes. In some embodiments, the fungal celloverexpresses at least one homologous and/or heterologous gene encodinga saccharide hydrolysis enzyme (e.g., beta-glucosidase). It is notintended that the present invention be limited to any particularenzyme(s), as numerous enzymes find use in the present invention. Insome embodiments, the enzyme is any one of a variety of endoglucanases,cellobiohydrolases, beta-glucosidases, endoxylanases, beta-xylosidases,arabinofuranosidases, alpha-glucuronidases, acetylxylan esterases,feruloyl esterases, alpha-glucuronyl esterases, and/or any other enzymeinvolved in saccharide hydrolysis. In some embodiments, the fungal cellis genetically modified to increase expression of beta-glucosidase.Thus, in some embodiments, a fungal cell comprises a polynucleotidesequence for increased expression of beta-glucosidase-encodingpolynucleotide and further can be genetically modified to deletepolynucleotides encoding one or more endogenous glucose and/orcellobiose oxidizing enzymes.

In some embodiments, the saccharide hydrolysis enzyme is endogenous tothe fungal cell. In some embodiments, the saccharide hydrolysis enzymeis exogenous to the fungal cell. In some other embodiments, the enzymemixture further comprises a saccharide hydrolysis enzyme that isheterologous to the fungal cell. Still further, in some embodiments, theprocess for generating glucose comprises contacting cellulose with anenzyme mixture that comprises a saccharide hydrolysis enzyme that isheterologous to the fungal cell.

In some embodiments, the fungal cells of the present invention aregenetically modified to increase the expression of a saccharidehydrolysis enzyme using any of a variety of methods that are known tothose of skill in the art. In some embodiments, the hydrolysisenzyme-encoding polynucleotide sequence is adapted for increasedexpression in a host fungal cell.

EXPERIMENTAL

The following examples, including experiments and results achieved, areprovided for illustrative purposes only and are not to be construed aslimiting the present invention.

In the experimental disclosure below, the following abbreviations apply:ppm (parts per million); M (molar); mM (millimolar), uM and μM(micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg(milligrams); ug and μg (micrograms); L and l (liter); ml and mL(milliliter); cm (centimeters); mm (millimeters); um and μm(micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s)(hour(s)); U (units); MW (molecular weight); rpm (rotations per minute);° C. (degrees Centigrade); DNA (deoxyribonucleic acid); RNA (ribonucleicacid); HPLC (high pressure liquid chromatography); MES (2-N-morpholinoethanesulfonic acid); FIOPC (fold improvements over positive control);YPD (10 g/L yeast extract, 20 g/L peptone, and 20 g/L dextrose); SOE-PCR(splicing by overlapping extension PCR); ARS (ARS Culture Collection orNRRL Culture Collection, Peoria, Ill.); Axygen (Axygen, Inc., UnionCity, Calif.); Lallemand (Lallemand Ethanol Technology, Milwaukee,Wis.); Dual Biosystems (Dual Biosystems AG, Schlieven, Switzerland);Alphalyse (Alphalyse Inc., Palo Alto, Calif.); Dyadic (DyadicInternational, Inc., Jupiter, Fla.); Promega (Promega, Inc., Madison,Wis.); Megazyme (Megazyme International Ireland, Ltd., Wicklow,Ireland); McMaster (McMaster Regional Centre for Mass Spectrometry inHamilton, Ontario, Canada); Sigma-Aldrich (Sigma-Aldrich, St. Louis,Mo.); Dasgip (Dasgip Biotools, LLC, Shrewsbury, Mass.); Difco (DifcoLaboratories, BD Diagnostic Systems, Detroit, Mich.); PCRdiagnostics(PCRdiagnostics, by E coli SRO, Slovak Republic); Agilent (AgilentTechnologies, Inc., Santa Clara, Calif.); Molecular Devices (MolecularDevices, Sunnyvale, Calif.); Symbio (Symbio, Inc., Menlo Park, Calif.);Sartorius (Sartorius AG, Goettingen, Germany); Finnzymes (part of ThermoFisher Scientific, Lafayette, Colo.); Dionex (now part of Thermo FisherScientific, Lafayette, Colo.); Idex (Idex Health and Science Group, OakHarbor, Wash.); Microbeads (Microbeads A/S, Skedsmokorset, Norway);Calbiochem (Calbiochem-Novabiochem International, Inc., La Jolla,Calif.); Newport (Newport Scientific, Australia); and Bio-Rad (Bio-RadLaboratories, Hercules, Calif.).

The following polynucleotide and polypeptide sequences find use in thepresent invention. As shown below, the polynucleotide sequence isfollowed by the encoded polypeptide.

M. thermophila GO1: (SEQ ID NO: 1)ATGGGCTTCCTCGCCGCCACTCTTGTGTCCTGTGCCGCTCTCGCGAGCGCAGCAAGCATCCCACGTCCCCATGCCAAGCGCCAGGTCTCCCAGCTTCGCGACGATTATGACTTCGTGATCGTTGGCGGTGGAACTAGCGGCCTCACTGTAGCCGATCGGCTGACAGAGGCCTTTCCAGCCAAGAACGTCCTTGTCATTGAGTATGGAGACGTCCACTACGCCCCGGGAACCTTCGATCCGCCGACGGACTGGATCACACCTCAGCCTGATGCCCCCCCTTCCTGGTCTTTCAATTCCCTCCCCAACCCAGACATGGCAAACACAACAGCGTTTGTGCTAGCCGGCCAAGTGGTGGGTGGAAGCAGTGCCGTGAACGGCATGTTCTTTGACCGCGCATCCCGCCACGACTACGATGCGTGGACCGCGGTCGGCGGGTCCGGGTTCGAACAGTCCAGCCACAAGTGGGACTGGGAGGGGCTGTTCCCTTTCTTCCAGAAGAGCGTCACGTTCACGGAACCGCCGGCCGACATCGTCCAGAAGTATCACTACACCTGGGACCTGTCTGCCTACGGCAATGGCTCAACCCCCATCTACAGCAGCTATCCGGTCTTCCAGTGGGCCGACCAGCCGTTACTTAACCAGGCATGGCAGGAGATGGGAATCAATCCGGTGACCGAATGCGCCGGCGGCGACAAGGAGGGTGTCTGCTGGGTTCCCGCCTCGCAGCACCCTGTCACGGCGAGGAGGTCGCACGCCGGGCTCGGCCACTACGCCGATGTGCTCCCGCGAGCCAATTACGACCTCCTCGTTCAACACCAGGTTGTCAGGGTAGTATTCCCCAATGGGCCGAGCCACGGACCGCCGCTTGTCGAGGCGCGGTCCCTGGCCGACAACCACCTGTTCAACGTGACTGTGAAGGGCGAAGTCATCATCTCGGCGGGCGCTCTGCACACCCCGACCGTCCTTCAACGGAGCGGCATCGGCCCGGCATCCTTCTTGGACGACGCCGGGATCCCCGTGACGCTTGACCTGCCGGGCGTCGGCGCCAACCTCCAGGACCACTGCGGTCCGCCCGTCACGTGGAACTACACCGAGCCCTACACCGGCTTCTTCCCGCTCCCCTCCGAGATGGTCAACAACGCGACCTTCAAAGCCGAAGCCATCACCGGCTTCGACGAGGTCCCGGCCCGCGGCCCCTACACGCTCGCCGGGGGCAACAACGCCATCTTCGTATCGCTCCCACACCTCACGGCCGACTACGGCGCCATCACCGCAAATATCCGCGCCATGGTCGCCGACGGAACCGCCGCCTCCTATCTCGCGGCCGACGTCCGCACCATCCCGGGGATGGTGGCCGGCTACGAGGCCCAGCTCCTCGTGCTCGCCGACCTGCTCGACAACCCGGAGGCGCCCAGCCTGGAGACGCCGTGGGCGACGAGCGAGGCGCCGCAGACGTCGTCGGTCCTGGCCTTCCTGCTGCACCCGCTCAGCCGCGGCAGCGTGCGGCTCAACCTCAGCGACCCGCTCGCGCAGCCCGTGCTCGACTACCGCTCCGGGTCCAACCCGGTCGACATCGACCTGCACCTCGCCCACGTGCGCTTCCTGCGCGGCCTGCTCGACACGCCCACCATGCAGGCCCGCGGGGCGCTCGAGACGGCCCCCGGCTCGGCCGTGGCCGACAGCGACGAGGCGCTGGGGGAGTACGTGCGCTCGCACAGCACGCTGTCCTTCATGCACCCGTGCTGCACGGCCGCCATGCTGCCCGAGGACCGGGGCGGCGTCGTCGGGCCGGACCTCAAGGTGCACGGGGCCGAGGGCCTGAGGGTCGTGGACATGAGCGTGATGCCGCTGTTGCCGGGGGCGCACCTGAGCGCCACTGCTTATGCGGTGGGGGAGAAAGCTGCGGATATTATCATCCAGGAGTGGATGGACAAGGAGCAGTGA (SEQ ID NO: 2)MGFLAATLVSCAALASAASIPRPHAKRQVSQLRDDYDFVIVGGGTSGLTVADRLTEAFPAKNVLVIEYGDVHYAPGTFDPPTDWITPQPDAPPSWSFNSLPNPDMANTTAFVLAGQVVGGSSAVNGMFFDRASRHDYDAWTAVGGSGFEQSSHKWDWEGLFPFFQKSVTFTEPPADIVQKYHYTWDLSAYGNGSTPIYSSYPVFQWADQPLLNQAWQEMGINPVTECAGGDKEGVCWVPASQHPVTARRSHAGLGHYADVLPRANYDLLVQHQVVRVVFPNGPSHGPPLVEARSLADNHLFNVTVKGEVIISAGALHTPTVLQRSGIGPASFLDDAGIPVTLDLPGVGANLQDHCGPPVTWNYTEPYTGFFPLPSEMVNNATFKAEAITGFDEVPARGPYTLAGGNNAIFVSLPHLTADYGAITANIRAMVADGTAASYLAADVRTIPGMVAGYEAQLLVLADLLDNPEAPSLETPWATSEAPQTSSVLAFLLHPLSRGSVRLNLSDPLAQPVLDYRSGSNPVDIDLHLAHVRFLRGLLDTPTMQARGALETAPGSAVADSDEALGEYVRSHSTLSFMHPCCTAAMLPEDRGGVVGPDLKVHGAEGLRVVDMSVMPLLPGAHLSATAYAVGEKAADIIIQEWMDKEQ M. thermophila GO2: (SEQ ID NO: 3)ATGGAGCTGCTTCGAGTCTCCCTCGCCGCTGTTGCACTCTCCCCATTAATATTATTCGGCGTTGCAGCCGCCCACCCTACCGCCCGATCCATTGCCCGCTCCACGATTCTTGACGGAGCCGATGGCCTTCTTCCGGAGTATGACTACATCATCATCGGGGGCGGCACGTCCGGATTGACTGTCGCCGACAGACTCACGGAGAATAGAAAGCGCAAGTTTTCCCGCTCTCCCCTCCCAACGTCACCCGCCCGATCGTCACCGGCGTGGTGTTATTCTGTTCTTGTTTTGGAAAGAGGCATTTTCCAGAACTCTAGCTCGGTGACCACCATTTCTGGGGGAAGCAGAGGCCTCTTCGATCCAAGTCTGACCTTCAACATCAACTCCGTTCCCCAAGCTGGGCTGGACAACCGCAGCATTGCCGTCATTGGCGGGTTGATCCTCGGCGGCAGCTCCGGCGTCAACGGGCTTCAAGTCCTCCGTGGACAAAGAGAAGACTATGACCGCTGGGGATCGTACTTTGGGCCAAACTCTGACTGGAGTTGGAAAGGTCTCCTGCCGTATTTCAAGAAGGCATGGAATTTCCATCCGCCCAGGCCAGAGCTGGTCAGTCAGTTCGACATCAAGTACGACCCCAGCTACTGGGGCAACACGTCTGACGTGCACGCATCTTTCCCAACCACTTTCTGGCCGGTGCTCAAATTGGAGATGGCTGCATTTGGTGACATCCCTGGGGTCGAATATCCGCCCGACTCTGCTTCTGGCGAGACCGGGGCGTATTGGCACCCAGCGTCCGTTGACCCAGCGACAGTCCTCCGCTCCTTCGCTCGGCCCGCGCATTGGGACAACATTGAGGCGGCACGTCCCAATTACCACACCCTGACCGGGCAACGCGTATTGAAGGTCGCATTTGATGGCAATCGAGCGACCAGCGTCGTCTTCGTGCCGGCGAATGCAACGGATCACAGCACTGCCAGGTCCGTGAAGGCCAAGAAGGAGATCGTCTTGGCCGCCGGCGCCATTCACACGCCCCAAATCCTACAGGCGAGCGGAGTAGGGCCGAAGCAGGTCCTGAAGGAAGCAGGCGTGCCGCTTGTCGTTGACGCTCCCGGTGTCGGCAGCAATTTCCAAGACCAGCCGTATGTGGTTGCTCCCACCTTCAATTTTACCAAGTTCCCCTTCCACCCGGACTTCTACGACATGATTCTGAACCAGACTTTTATCGCCGAGGCTCAGGCCCAGTTTGAAAAGGACCGTACCGGACCTCACACCATCGCATCCGGCTATTGCGGCAGCTGGCTCCCCCTCCAGATCATTGCCCCAAATTCGTGGAAGGACATCGCTAGGCGGTACGAATCCCAAGACCCAGCCGCCTACCTCCCCGCCGGCACCGATGAGACCGTCATCGAGGGGTACAGGGCGCAGCAGAAAGCACTAGCGAGGTCCATGAGGAGCAAGCAATCGGCAATGTATAACTTCTTCCTGAGGGGCGGCTACGAAGAGGGTTCTGTCGTCTACTTGCACCCAACCAGCCGTGGCACCGTTCGCATCAACCGATCCGACCCCTTCTTCTCGCCGCCCGAGGTCGACTACAGGGCACTGAGCAACCCTACCGACCTGGAGGTCCTGCTCGAATTCACTCCCTTCACCCGCAGGTACTTCTTGGAGACGAGGTTGAAGTCCCTCGACCCGGTCGAGCTGTCGCCCGGTGCCAACGTCACGGCGCCCGCCGACATCGAGGCCTGGCTTCGCAGCGTCATGATCCCGTCCTCCTTCCATCCCATCGGCACGGCCGCCATGTTGCCTAGGCACCTCGGTGGTGTCGTGGACGAGAACCTTCTGGTGTACGGGGTCGAAGGCTTGAGTGTCGTCGACGCCAGCGTCATGCCCGACTTGCCGGGCTCATACACGCAGCAGACCGTGTATGCTATTGCTGAGAAGGCCGCGGATCTCATTAAGAGCAGGG CTTGA(SEQ ID NO: 4)MELLRVSLAAVALSPLILFGVAAAHPTARSIARSTILDGADGLLPEYDYIIIGGGTSGLTVADRLTENRKRKFSRSPLPTSPARSSPAWCYSVLVLERGIFQNSSSVTTISGGSRGLFDPSLTFNINSVPQAGLDNRSIAVIGGLILGGSSGVNGLQVLRGQREDYDRWGSYFGPNSDWSWKGLLPYFKKAWNFHPPRPELVSQFDIKYDPSYWGNTSDVHASFPTTFWPVLKLEMAAFGDIPGVEYPPDSASGETGAYWHPASVDPATVLRSFARPAHWDNIEAARPNYHTLTGQRVLKVAFDGNRATSVVFVPANATDHSTARSVKAKKEIVLAAGAIHTPQILQASGVGPKQVLKEAGVPLVVDAPGVGSNFQDQPYVVAPTFNFTKFPFHPDFYDMILNQTFIAEAQAQFEKDRTGPHTIASGYCGSWLPLQIIAPNSWKDIARRYESQDPAAYLPAGTDETVIEGYRAQQKALARSMRSKQSAMYNFFLRGGYEEGSVVYLHPTSRGTVRINRSDPFFSPPEVDYRALSNPTDLEVLLEFTPFTRRYFLETRLKSLDPVELSPGANVTAPADIEAWLRSVMIPSSFHPIGTAAMLPRHLGGVVDENLLVYGVEGLSVVDASVMPDLPGSYTQQTVYAIAEKAADLIKSRA M. thermophila CDH1: (SEQ ID NO: 5)ATGAGGACCTCCTCTCGTTTAATCGGTGCCCTTGCGGCGGCACTCTTGCCGTCTGCCCTTGCGCAGAACAACGCGCCGGTAACCTTCACCGACCCGGACTCGGGCATTACCTTCAACACGTGGGGTCTCGCCGAGGATTCTCCCCAGACTAAGGGCGGTTTCACTTTTGGTGTTGCTCTGCCCTCTGATGCCCTCACGACAGACGCCAAGGAGTTCATCGGTTACTTGAAATGCGCGAGGAACGATGAGAGCGGTTGGTGCGGTGTCTCCCTGGGCGGCCCCATGACCAACTCGCTCCTCATCGCGGCCTGGCCCCACGAGGACACCGTCTACACCTCTCTCCGCTTCGCCACCGGCTATGCCATGCCGGATGTCTACCAGGGGGACGCCGAGATCACCCAGGTCTCCTCCTCTGTCAACTCGACGCACTTCAGCCTCATCTTCAGGTGCGAGAACTGCCTGCAATGGAGTCAAAGCGGCGCCACCGGCGGTGCCTCCACCTCGAACGGCGTGTTGGTCCTCGGCTGGGTCCAGGCATTCGCCGACCCCGGCAACCCGACCTGCCCCGACCAGATCACCCTCGAGCAGCACGACAACGGCATGGGTATCTGGGGTGCCCAGCTCAACTCCGACGCCGCCAGCCCGTCCTACACCGAGTGGGCCGCCCAGGCCACCAAGACCGTCACGGGTGACTGCGGCGGTCCCACCGAGACCTCTGTCGTCGGTGTCCCCGTTCCGACGGGCGTCTCGTTCGATTACATCGTCGTGGGCGGCGGTGCCGGTGGCATCCCCGCCGCCGACAAGCTCAGCGAGGCCGGCAAGAGTGTGCTGCTCATCGAGAAGGGCTTTGCCTCGACCGCCAACACCGGAGGCACTCTCGGCCCCGAGTGGCTCGAGGGCCACGACCTTACCCGCTTTGACGTGCCGGGTCTGTGCAACCAGATCTGGGTTGACTCCAAGGGGATCGCTTGCGAGGATACCGACCAGATGGCTGGCTGTGTCCTCGGCGGCGGTACCGCCGTGAATGCCGGCCTGTGGTTCAAGCCCTACTCGCTCGACTGGGACTACCTCTTCCCTAGTGGTTGGAAGTACAAAGACGTCCAGCCGGCCATCAACCGCGCCCTCTCGCGCATCCCGGGCACCGATGCTCCCTCGACCGACGGCAAGCGCTACTACCAACAGGGCTTCGACGTCCTCTCCAAGGGCCTGGCCGGCGGCGGCTGGACCTCGGTCACGGCCAATAACGCGCCAGACAAGAAGAACCGCACCTTCTCCCATGCCCCCTTCATGTTCGCCGGCGGCGAGCGCAACGGCCCGCTGGGCACCTACTTCCAGACCGCCAAGAAGCGCAGCAACTTCAAGCTCTGGCTCAACACGTCGGTCAAGCGCGTCATCCGCCAGGGCGGCCACATCACCGGCGTCGAGGTCGAGCCGTTCCGCGACGGCGGTTACCAAGGCATCGTCCCCGTCACCAAGGTTACGGGCCGCGTCATCCTCTCTGCCGGTACCTTTGGCAGTGCAAAGATCCTGCTGAGGAGCGGTATCGGTCCGAACGATCAGCTGCAGGTTGTCGCGGCCTCGGAGAAGGATGGCCCTACCATGATCAGCAACTCGTCCTGGATCAACCTGCCTGTCGGCTACAACCTGGATGACCACCTCAACACCGACACTGTCATCTCCCACCCCGACGTCGTGTTCTACGACTTCTACGAGGCGTGGGACAATCCCATCCAGTCTGACAAGGACAGCTACCTCAACTCGCGCACGGGCATCCTCGCCCAAGCCGCTCCCAACATTGGGCCTATGTTCTGGGAAGAGATCAAGGGTGCGGACGGCATTGTTCGCCAGCTCCAGTGGACTGCCCGTGTCGAGGGCAGCCTGGGTGCCCCCAACGGCAAGACCATGACCATGTCGCAGTACCTCGGTCGTGGTGCCACCTCGCGCGGCCGCATGACCATCACCCCGTCCCTGACAACTGTCGTCTCGGACGTGCCCTACCTCAAGGACCCCAACGACAAGGAGGCCGTCATCCAGGGCATCATCAACCTGCAGAACGCCCTCAAGAACGTCGCCAACCTGACCTGGCTCTTCCCCAACTCGACCATCACGCCGCGCCAATACGTTGACAGCATGGTCGTCTCCCCGAGCAACCGGCGCTCCAACCACTGGATGGGCACCAACAAGATCGGCACCGACGACGGGCGCAAGGGCGGCTCCGCCGTCGTCGACCTCAACACCAAGGTCTACGGCACCGACAACCTCTTCGTCATCGACGCCTCCATCTTCCCCGGCGTGCCCACCACCAACCCCACCTCGTACATCGTGACGGCGTCGGAGCACGCCTCGGCCCGCATCCTCGCCCTGCCCGACCTCACGCCCGTCCCCAAGTACGGGCAGTGCGGCGGCCGCGAATGGAGCGGCAGCTTCGTCTGCGCCGACGGCTCCACGTGCCAGATGCAGAACGAGTGGTACTCGCAGTGCTTGTGA (SEQ ID NO: 6)MRTSSRLIGALAAALLPSALAQNNAPVTFTDPDSGITFNTWGLAEDSPQTKGGFTFGVALPSDALTTDAKEFIGYLKCARNDESGWCGVSLGGPMTNSLLIAAWPHEDTVYTSLRFATGYAMPDVYQGDAEITQVSSSVNSTHFSLIFRCENCLQWSQSGATGGASTSNGVLVLGWVQAFADPGNPTCPDQITLEQHDNGMGIWGAQLNSDAASPSYTEWAAQATKTVTGDCGGPTETSVVGVPVPTGVSFDYIVVGGGAGGIPAADKLSEAGKSVLLIEKGFASTANTGGTLGPEWLEGHDLTRFDVPGLCNQIWVDSKGIACEDTDQMAGCVLGGGTAVNAGLWFKPYSLDWDYLFPSGWKYKDVQPAINRALSRIPGTDAPSTDGKRYYQQGFDVLSKGLAGGGWTSVTANNAPDKKNRTFSHAPFMFAGGERNGPLGTYFQTAKKRSNFKLWLNTSVKRVIRQGGHITGVEVEPFRDGGYQGIVPVTKVTGRVILSAGTFGSAKILLRSGIGPNDQLQVVAASEKDGPTMISNSSWINLPVGYNLDDHLNTDTVISHPDVVFYDFYEAWDNPIQSDKDSYLNSRTGILAQAAPNIGPMFWEEIKGADGIVRQLQWTARVEGSLGAPNGKTMTMSQYLGRGATSRGRMTITPSLTTVVSDVPYLKDPNDKEAVIQGIINLQNALKNVANLTWLFPNSTITPRQYVDSMVVSPSNRRSNHWMGTNKIGTDDGRKGGSAVVDLNTKVYGTDNLFVIDASIFPGVPTTNPTSYIVTASEHASARILALPDLTPVPKYGQCGGREWSGSFVCADGSTCQMQNEWYSQCL M. thermophila CDH2:(SEQ ID NO: 7) ATGAAGCTACTCAGCCGCGTTGGGGCGACCGCCCTAGCGGCGACGTTGTCACTGCAGCAATGTGCAGCCCAGATGACCGAGGGGACCTACACCGATGAGGCTACCGGTATCCAATTCAAGACGTGGACCGCCTCCGAGGGCGCCCCTTTCACGTTTGGCTTGACCCTCCCCGCGGACGCGCTGGAAAAGGATGCCACCGAGTACATTGGTCTCCTGCGTTGCCAAATCACCGATCCCGCCTCGCCCAGCTGGTGCGGTATCTCCCACGGCCAGTCCGGCCAGATGACGCAGGCGCTGCTGCTGGTCGCCTGGGCCAGCGAGGACACCGTCTACACGTCGTTCCGCTACGCCACCGGCTACACGCTCCCCGGCCTCTACACGGGCGACGCCAAGCTGACCCAGATCTCCTCCTCGGTCAGCGAGGACAGCTTCGAGGTGCTGTTCCGCTGCGAAAACTGCTTCTCCTGGGACCAGGATGGCACCAAGGGCAACGTCTCGACCAGCAACGGCAACCTGGTCCTCGGCCGCGCCGCCGCGAAGGATGGTGTGACGGGCCCCACGTGCCCGGACACGGCCGAGTTCGGTTTCCATGATAACGGTTTCGGACAGTGGGGTGCCGTGCTTGAGGGTGCTACTTCGGACTCGTACGAGGAGTGGGCTAAGCTGGCCACGACCACGCCCGAGACCACCTGCGATGGCACTGGCCCCGGCGACAAGGAGTGCGTTCCGGCTCCCGAGGACACGTATGATTACATCGTTGTCGGTGCCGGCGCCGGTGGTATCACCGTCGCCGACAAGCTCAGCGAGGCCGGCCACAAGGTCCTTCTCATCGAGAAGGGACCCCCTTCGACCGGCCTGTGGAACGGGACCATGAAGCCCGAGTGGCTCGAGAGCACCGACCTTACCCGCTTCGACGTTCCCGGCCTGTGCAACCAGATCTGGGTCGACTCTGCCGGCATCGCCTGCACCGATACCGACCAGATGGCGGGCTGCGTTCTCGGCGGTGGCACCGCTGTCAACGCTGGTTTGTGGTGGAAGCCCCACCCCGCTGACTGGGATGAGAACTTCCCCGAAGGGTGGAAGTCGAGCGATCTCGCGGATGCGACCGAGCGTGTCTTCAAGCGCATCCCCGGCACGTCGCACCCGTCGCAGGACGGCAAGTTGTACCGCCAGGAGGGCTTCGAGGTCATCAGCAAGGGCCTGGCCAACGCCGGCTGGAAGGAAATCAGCGCCAACGAGGCGCCCAGCGAGAAGAACCACACCTATGCACACACCGAGTTCATGTTCTCGGGCGGTGAGCGTGGCGGCCCCCTGGCGACGTACCTTGCCTCGGCTGCCGAGCGCAGCAACTTCAACCTGTGGCTCAACACTGCCGTCCGGAGGGCCGTCCGCAGCGGCAGCAAGGTCACCGGCGTCGAGCTCGAGTGCCTCACGGACGGTGGCTTCAGCGGGACCGTCAACCTGAATGAGGGCGGTGGTGTCATCTTCTCGGCCGGCGCTTTCGGCTCGGCCAAGCTGCTCCTTCGCAGCGGTATCGGTCCTGAGGACCAGCTCGAGATTGTGGCGAGCTCCAAGGACGGCGAGACCTTCACTCCCAAGGACGAGTGGATCAACCTCCCCGTCGGCCACAACCTGATCGACCATCTCAACACTGACCTCATTATCACGCACCCGGATGTCGTTTTCTATGACTTCTATGCGGCCTGGGACGAGCCCATCACGGAGGATAAGGAGGCCTACCTGAACTCGCGGTCCGGCATTCTCGCCCAGGCGGCGCCCAATATCGGCCCTATGATGTGGGATCAAGTCACGCCGTCCGACGGCATCACCCGCCAGTTCCAGTGGACATGCCGTGTTGAGGGCGACAGCTCCAAGACCAACTCGACCCACGCCATGACCCTCAGCCAGTACCTCGGCCGTGGCGTCGTCTCGCGCGGCCGGATGGGCATCACCTCCGGGCTGAGCACGACGGTGGCCGAGCACCCGTACCTGCACAACAACGGCGACCTGGAGGCGGTCATCCAGGGGATCCAGAACGTGGTGGACGCGCTCAGCCAGGTGGCCGACCTCGAGTGGGTGCTCCCGCCGCCCGACGGGACGGTGGCCGACTACGTCAACAGCCTGATCGTCTCGCCGGCCAACCGCCGGGCCAACCACTGGATGGGCACGGCCAAGCTGGGCACCGACGACGGCCGCTCGGGCGGCACCTCGGTCGTCGACCTCGACACCAAGGTGTACGGCACCGACAACCTGTTCGTCGTCGACGCGTCCGTCTTCCCCGGCATGTCGACGGGCAACCCGTCGGCCATGATCGTCATCGTGGCCGAGCAGGCGGCGCAGCGCATCCTGGCCCTGCGGTCTTAA (SEQ ID NO: 8)MKLLSRVGATALAATLSLQQCAAQMTEGTYTDEATGIQFKTWTASEGAPFTFGLTLPADALEKDATEYIGLLRCQITDPASPSWCGISHGQSGQMTQALLLVAWASEDTVYTSFRYATGYTLPGLYTGDAKLTQISSSVSEDSFEVLFRCENCFSWDQDGTKGNVSTSNGNLVLGRAAAKDGVTGPTCPDTAEFGFHDNGFGQWGAVLEGATSDSYEEWAKLATTTPETTCDGTGPGDKECVPAPEDTYDYIVVGAGAGGITVADKLSEAGHKVLLIEKGPPSTGLWNGTMKPEWLESTDLTRFDVPGLCNQIWVDSAGIACTDTDQMAGCVLGGGTAVNAGLWWKPHPADWDENFPEGWKSSDLADATERVFKRIPGTSHPSQDGKLYRQEGFEVISKGLANAGWKEISANEAPSEKNHTYAHTEFMFSGGERGGPLATYLASAAERSNFNLWLNTAVRRAVRSGSKVTGVELECLTDGGFSGTVNLNEGGGVIFSAGAFGSAKLLLRSGIGPEDQLEIVASSKDGETFTPKDEWINLPVGHNLIDHLNTDLIITHPDVVFYDFYAAWDEPITEDKEAYLNSRSGILAQAAPNIGPMMWDQVTPSDGITRQFQWTCRVEGDSSKTNSTHAMTLSQYLGRGVVSRGRMGITSGLSTTVAEHPYLHNNGDLEAVIQGIQNVVDALSQVADLEWVLPPPDGTVADYVNSLIVSPANRRANHWMGTAKLGTDDGRSGGTSVVDLDTKVYGTDNLFVVDASVFPGMSTGNPSAMIVIVAEQAAQRILALRSAspergillus oryzae pyranose oxidase: (SEQ ID NO: 9)ATGTCCATGACATCAGGACGTCAAGCGTTTACTTCCGAGTGCAGAGATTCAAATACCACAAATTCATTTTGGTTGGCTAATTCACCGACTCTCACACTTGGCTCTACGATGCAGGTCGTGGGGTCCGGCCCCATCGGCGCCACCTATGCCAAGATTCTAGCTGACGCCGGCAAGGATGTCCTCATGGTTGAGACTGGCACCCAGGAAAGTAAGATTGCTGGAGAGCATAAGAAGAATGCTATCAACTACCAGAAAGATATCGATGCCTTTGTGCATGTCATTAAGGTAATCAGCTCAAGAATTAGCACCTTTGAGTGTATTTCTCTAACTTTCGATCTTCTCCTCTTTCAGGGAAGTCTACACTACACGTCTGTACCGACCAACAAAGCCGCCGTTCCTACACTGGCTCCGATCTCCTGGAAAGCGAACGGCCAAATTTTCAACGGACAGAATCCCCGCCAGGATCCAAACGTAAACCTGGATGCCAATGGTGTGGCACGTAATGTGGGCGGCATGTCTACCCACTGGACTTGTGCGACTCCCCGACAGAAAGAGAAGGTTGAACGCAGCGATATATTCAGTGGTGACGAATGGGATAGCCTGTACAAGGAGGCAGAAAAGTTGATCGGAACCAGCAAGACTGTGCTGAATGACTCGATCCGGCAAGAATTGGTCATGGAGATTCTGAATGACGAGTACGGGAAGCGATCAGCCGAACCACTACCTTTGGCTGCAAAGAGGAATGGCAATACGGCCTACATCACTTGGTCATCCTCGTCAACTATCCTTGACGCGATGAACTGTAAGAAGAAATTTACACTATGGCCCGAGCACCACTGTGAGAAGTTTAAAGTCGAGGAAACAGATAACGGGCCACAGGTCACCAAGGCTATAATCCGCAAACTCGCCACAGATAAACTGATTACAGTTAAGGCGAAAGTATTTATCGCTTGCGGGGGGCCTATACTTACACCCCAGCTACTTTTCAATTCGGGCTTCGTGCCGACAAAGCCCAACAGGGATCCCAGAACCCAAATACCATTAGAAGACGACGAGAAAGGCATCCCACCTCCACCGGATACTCTGGAGCATCTCAAGCTTCCTGCTCTAGGACGCTATCTGACAGAGCAAAGCATGTGCTTCTGCCAAATTGTTCTGAAAAAAGAATGGATTGAGGCAGTGGCTAATCCAAAAAAGAACCCTTATCAAAGCGATGGGGTGAAACGCAAAAAGTGGGAGAAGCTCAAGGAAGGGTGGAAGGAAAGGGTCCAGGAACATATGAAAAGGTTTAATGACCCTATTCCCTTCCCGTTCGATGATTTGGACCCTCAGGTTACTCTACCCTTGGACTATCACCATCCGTGGCATACCCAAATCCATCGCGATGCCTTCTCCTATGGCGCAGCACCCCCAGCCATTGATAAGCGGACCATTGTTGACCTCCGATTCTTCGGAACGGTTGAGCCGGACTGGAAGAACTATGTGACCTTTGAAACCGACATCAGGGATGCGTACGGCATGCCCCAGCCCACCTTCCGCTACAAGCTGAACGATGAGGATCGCAAACGGTCGCACCAGATGATGAAAGATATGGAAGAGGCCGCTGGTGCTCTGGGTGGCTACCTCCCAGGGTCGGAGCCTCAATTTCTAGCTCCTGGCCTTGCACTGCACGTCTGTGGTACCACTAGAGCTCAGAAGAAGGAGAAAGAGTGTGACCCTGATCCCAAAGAGACCTCGTGCTGCGATGAGAACTCCAAGATCTGGGGTATCCACAACCTGTACGTGGGTGGGTTAAATGTGATCCCTGGTGCCAATGGGTCCAACCCTACCTTGACAGCAATGTGCTTCGCCATCAAAAGCGCGAAGAGTATCCTTGAAGGGAATTCTTAG (SEQ ID NO: 10)MSMTSGRQAFTSECRDSNTTNSFWLANSPTLTLGSTMQVVGSGPIGATYAKILADAGKDVLMVETGTQESKIAGEHKKNAINYQKDIDAFVHVIKVISSRISTFECISLTFDLLLFQGSLHYTSVPTNKAAVPTLAPISWKANGQIFNGQNPRQDPNVNLDANGVARNVGGMSTHWTCATPRQKEKVERSDIFSGDEWDSLYKEAEKLIGTSKTVLNDSIRQELVMEILNDEYGKRSAEPLPLAAKRNGNTAYITWSSSSTILDAMNCKKKFTLWPEHHCEKFKVEETDNGPQVTKAIIRKLATDKLITVKAKVFIACGGPILTPQLLFNSGFVPTKPNRDPRTQIPLEDDEKGIPPPPDTLEHLKLPALGRYLTEQSMCFCQIVLKKEWIEAVANPKKNPYQSDGVKRKKWEKLKEGWKERVQEHMKRFNDPIPFPFDDLDPQVTLPLDYHHPWHTQIHRDAFSYGAAPPAIDKRTIVDLRFFGTVEPDWKNYVTFETDIRDAYGMPQPTFRYKLNDEDRKRSHQMMKDMEEAAGALGGYLPGSEPQFLAPGLALHVCGTTRAQKKEKECDPDPKETSCCDENSKIWGIHNLYVGGLNVIPGANGSNPTLTAMCFAIKSAKSIL EGNSAcremonium strictum glucooligosaccharide oxidase: (SEQ ID NO: 11)ATGGTGCGCATCCAAGAGCTCACCGCGGCCTTGAGCCTCGCCTCAGTGGTCCAGGCTTCATGGATCCAGAAGCGCAACTCAATCAACGCCTGTCTCGCCGCCGCCGACGTCGAGTTCCACGAGGAAGACTCTGAAGGCTGGGACATGGACGGCACAGCCTTCAACCTCCGCGTCGACTACGACCCAGCTGCCATTGCCATCCCTCGCTCCACCGAGGATATCGCTGCTGCTGTCCAGTGCGGTCTTGATGCTGGTGTGCAGATCTCCGCCAAGGGTGGTGGTCACAGTTACGGTTCTTATGGGTTCGGTGGTGAGGATGGTCATCTTATGTTGGAGCTGGATCGTATGTACCGTGTGTCGGTTGATGATAATAATGTGGCGACTATTCAGGGCGGTGCTCGTCTTGGATACACTGCTCTCGAGCTTCTTGACCAGGGTAACCGTGCACTTTCTCACGGTACTTGCCCTGCCGTCGGTGTCGGCGGTCACGTCCTCGGCGGTGGTTACGGTTTCGCAACCCACACCCACGGTCTGACCCTCGACTGGCTGATCGGCGCCACCGTCGTTCTCGCTGATGCCTCCATCGTGCACGTCTCCGAGACCGAGAACGCCGATCTCTTCTGGGCCCTCCGTGGCGGCGGCGGTGGTTTCGCCATCGTCTCCGAGTTCGAGTTCAACACCTTCGAGGCCCCCGAGATCATCACCACTTACCAGGTCACCACCACCTGGAACCGGAAGCAGCACGTTGCCGGTCTCAAGGCTCTCCAGGACTGGGCTCAGAACACCATGCCCAGGGAGCTCAGCATGCGTCTTGAGATCAACGCCAACGCTCTCAACTGGGAGGGTAACTTCTTCGGTAACGCCAAGGACCTCAAGAAGATTCTTCAGCCTATCATGAAGAAGGCGGGTGGCAAGTCTACCATTTCCAAGCTCGTTGAGACCGATTGGTATGGCCAGATCAACACCTACCTCTACGGTGCTGACTTGAACATCACCTACAACTACGACGTCCACGAGTACTTCTACGCCAACAGCTTGACCGCTCCCCGTCTCTCCGACGAAGCCATCCAAGCCTTCGTCGACTACAAGTTCGACAACTCCTCCGTCCGCCCCGGCCGCGGCTGGTGGATTCAATGGGACTTCCACGGCGGCAAGAACTCTGCCCTGGCCGCCGTCTCCAACGACGAAACCGCCTACGCCCACCGCGACCAGCTCTGGCTCTGGCAGTTCTACGACAGCATCTATGACTACGAGAACAACACCTCTCCCTACCCGGAGAGCGGTTTCGAGTTCATGCAGGGCTTCGTCGCTACCATCGAGGACACTCTCCCTGAGGACAGGAAGGGCAAGTACTTCAACTACGCCGACACCACGCTTACCAAGGAGGAGGCGCAGAAGTTGTACTGGAGGGGCAACCTTGAGAAGTTGCAGGCTATCAAGGCCAAGTACGATCCTGAGGATGTGTTTGGTAATGTTGTCTCTGTTGAGCCCATTGCCTAG (SEQ ID NO: 12)MVRIQELTAALSLASVVQASWIQKRNSINACLAAADVEFHEEDSEGWDMDGTAFNLRVDYDPAAIAIPRSTEDIAAAVQCGLDAGVQISAKGGGHSYGSYGFGGEDGHLMLELDRMYRVSVDDNNVATIQGGARLGYTALELLDQGNRALSHGTCPAVGVGGHVLGGGYGFATHTHGLTLDWLIGATVVLADASIVHVSETENADLFWALRGGGGGFAIVSEFEFNTFEAPEIITTYQVTTTWNRKQHVAGLKALQDWAQNTMPRELSMRLEINANALNWEGNFFGNAKDLKKILQPIMKKAGGKSTISKLVETDWYGQINTYLYGADLNITYNYDVHEYFYANSLTAPRLSDEAIQAFVDYKFDNSSVRPGRGWWIQWDFHGGKNSALAAVSNDETAYAHRDQLWLWQFYDSIYDYENNTSPYPESGFEFMQGFVATIEDTLPEDRKGKYFNYADTTLTKEEAQKLYWRGNLEKLQAIKAKYDPEDVFGNVVSVEPIAAgaricus bisporus pyranose dehydrogenase: (SEQ ID NO: 13)ATGATACCTCGAGTGGCCAAATTCAACTTTCGACTCTTGTCTCTCGCATTATTGGGGATTCAGGTTGCACGCAGTGCCATCACATACCAAAACCCGACCGATTTACCTGGTGACGTTGACTATGATTTCATCGTTGCTGGCGGTGGAACTGCAGGTTTAGTTGTGGCCTCTCGTCTCAGTGAGAATCCGGAATGGAATGTACTGGTCATCGAGGCCGGGCCTTCCAACAAGGACGTCTTCGAAACACGGGTCCCTGGCCTTTCTTCGGAACTCCGGCCACGTTTTGATTGGAATTATACAACGATTCCTCAAGATGCTCTCGGTGGCAGGAGCCTGAATTACTCGAGGGCGAAGCTCTTAGGCGGTTGCAGTAGCCATAATGGGATGGTTTACACACGATGTTCGAGAGACGATTGGGACAATTATGCCGAAATCACCGGTAATCAAGCATTTAGCTGGGACAGCATCCTACCTGTCATGAAGAGGGCTGAGAAATTCAGTAAAGATTCCTCTCATAAACCGGTAAAGGGCCATATTGACCCCTCCGTGCACGGTGGTGACGGAAAATTGTCCGTGGTCGCATCATACACCAACGCCTCTTTCAATGACTTATTACTTGAAACCGCGAAAGAATTAAGCGGTGAATTTCCGTTCAAATTGGATATGAATGACGGGCGGCCTCTTGGATTAACTTGGACTCAGTATACGATTGATCAACGCGGGGAGCGGAGCAGCTCTGCAACAGCGTATTTAGAGGGTACTGGAAATAACGTCCATGTCTTGGTTAACACTCTTGTTACCCGTATAGTCTCAGCAGAAAATGGGACCGACTTCCGAAGCGTCGAGTTTGCTACTGATGCCGACAGCCCAAAGATCCAATTACGAGCGAAAAAGGAAGTCATTGTATCTGGAGGAGTCATCAATTCGCCTCAGATCCTCATGAATTCCGGCATTGGGGGCCGAGAGGTGCTTGGAGCTAATGGAATTGACACATTGGTGGATAATCCGAGTGTCGGGAAAAATTTATCGGACCAGGCTGCAACAATTATAATGCTCGATACAACACTCCCTATTACTGATTATGATGTTGATGCAGCGCTTATTGAATGGAAGAAGTCGCACACTGGACCTCTAGCCCAAGGAGGTCGCCTAAACCACCTTACATGGGTACGATTGCCTGATGACAAGCTGGATGGACTTGATCCTTCAAGTGGCGAAAATTCGCCACATATTGAGTTCCAATTCGGGCAAATTAGCCACCAGCTCCCTCCCAGTGGTCTAACACGTTTTAGCTTCTATCGACACTGTTCTCCAATTCCGCCGTTGATCAACCTCTACACTGTTTCGCGGGGTTCTATTTCTCTCAGTAACAACGATCCGTTCTCCCACCCACTCATCGATCTCAACATGTTTGGAGAGGAAATAGATCCCGCTATTCTGCGTGAGGGTATTCGCAGTGCCCGAAGAATGCTTTCTTCCCAAGCATTCAAAGGCTTTGTCGGTGAAACGGTGTTTCCTCCAAGCGACGCTACCTCTGATGAAGATTTGGATACCTTCCTCAAAACGTCAACGTTTTCTTACGTGCATGGTGTGGGAACGTTGTCTATGTCTCCTCAGAGTGCCTCGTGGGGTGTCGTTAACCCTGATTTCCGTGTCAAAGGAACCAGTGGCCTGCGGGTTGTCGACGCGTCTGTGATTCCATTCGCTCCGGCGGGGCACACTCAAGAACCTGTTTATGCATTTGCTGAGCATGCAAGTGTGTTAATAGCGAAGAGCTACAGCTAA (SEQ ID NO: 14)MIPRVAKFNFRLLSLALLGIQVARSAITYQNPTDLPGDVDYDFIVAGGGTAGLVVASRLSENPEWNVLVIEAGPSNKDVFETRVPGLSSELRPRFDWNYTTIPQDALGGRSLNYSRAKLLGGCSSHNGMVYTRCSRDDWDNYAEITGNQAFSWDSILPVMKRAEKFSKDSSHKPVKGHIDPSVHGGDGKLSVVASYTNASFNDLLLETAKELSGEFPFKLDMNDGRPLGLTWTQYTIDQRGERSSSATAYLEGTGNNVHVLVNTLVTRIVSAENGTDFRSVEFATDADSPKIQLRAKKEVIVSGGVINSPQILMNSGIGGREVLGANGIDTLVDNPSVGKNLSDQAATIIMLDTTLPITDYDVDAALIEWKKSHTGPLAQGGRLNHLTWVRLPDDKLDGLDPSSGENSPHIEFQFGQISHQLPPSGLTRFSFYRHCSPIPPLINLYTVSRGSISLSNNDPFSHPLIDLNMFGEEIDPAILREGIRSARRMLSSQAFKGFVGETVFPPSDATSDEDLDTFLKTSTFSYVHGVGTLSMSPQSASWGVVNPDFRVKGTSGLRVVDASVIPFAPAGHTQEPVYAFAEHASVLIAKSYSTalaromyces stipitatus (ATCC 10500) glucose dehydrogenase:(SEQ ID NO: 15) ATGCGACTTGGCTCTATCGGCGCAGGCCTCGCTCTCCTCGCTGCCCTCGCTGTCCTCGCTGCCCACGTGCACGCCTTGGCACCGCGCACCCAGATTGCCGAGGAATACGATTTTGTCGTCGTTGGCGGCGGCCAGGCTGGTCTCGTGATCGGAGCTCGTCTGTCGGAGATTGCAAATTATACAGTTCTCGTGCTGGAGGCAGGGACGAATGGAGACGAATTTCGAGAACGAATAGGCACGTACAACTTTTATACTCCCGCATATTCCTACTACGAGTCACTATGGACGACACCAATGAATTGGGCATACTATACTGTGCCTCAATCCCATGCCGAGAATCGTCAAATTGAGTGGACCCGTGGTAAGGGGCTGGGCGGAAGTTCTGCGATCAACGGATTGTACCTGACTCGCCCCGGTAAAGAGGAGATCAATGCATGGAAAGACCTGCTAGGAGACATGGACGGGGCGGACAATTGGTCGTGGGATTCGTTCTATGCTGCAATGAAGAAGAGCGAGACTTTTACTCCCCCGTCGAATGAGATTGCTACAGAAGGGAACATTACATGGGACCTTTCTACTCGTGGTATTCAGGGACCGATTCAGGCAACGTATCCCGGCTATACCTTCCCCCAAGTCGGCGAATGGGTCATGTCTCTGGAAGCAATGGGCATTGCTAGTTCTAACGATATGTACGGTGGTGAGGTGTATGGCGCCGAAGTCTCGACGTCGAGTATCAATCCCACGAACTGGACACGCTCGTACAGCCGGACGGGATATCTCGACCCGCTCGCAGACAACGGCAATTACGACGTTGTGGCCGATGCGTTTGTCACGCGCATTCTCTTTGATGCTTCTTCTCCGTCGAATAATCTGACAGCAAACGGCGTGCAGTATACTCTTGACAACGGCAAGACAAACTGCACGGTCAAGGTCAAGAAAGAGGTGATCTTATCAGCTGGGACGGTTGGCAGTCCTGCGGTACTGCTCCACAGCGGTGTCGGTCCGAAAGATGTTCTTTCAGATGCTGGAGTTGAGCTGGTGTCTGAACTTCCTGGTGTGGGTCACCACCTTCAGGATCATTTTAACAACACCCTTTATCTCTCCTACATCGATTCAGCCATCGCCTACATCAATTCCACGCTGATGTACGGCGATAATCTGGACGCACTACAGAAGAACATCACCACTCAAATCAACCAATTCGTGCTGAACACGACTTACGATGCTGGTGTCATTGCAGGATACAAAGCAATTGCAAATATGACCGCAACCACAATCCTCAGTAGTTCTATCGGGCAAATTGAGCTCTTGTTCATGAATAGTGACTTAAACGGCGATATTGGTATCACTGCTGCTCTTCAACATCCTTACAGCCATGGACGCATATACATCAATTCCTCGAATCCGTTGGACTATCCCGTCATTGATCCGAATTATCTTGCTGTTTCTGCTGACTATGAAATCCTCCGCGACGGCCTCAATCTAGCCCGCCAACTCGGCAACACACAACCCCTAAGCAGCTGTCTAATAGCCGAAACAATCCCCGGTCCCAGCGTCAAAACCGACGACGACTGGCTTGAATGGATCCGCGAAGCGACGGGGACAGAGTTCCACCCTTCATCGTCCTGTGCGATGCTACCCCGAGAGCAAGGCGGAGTAGTCGATGCCAACCTGCGCGTCTACGGTCTTGCCAATGTTCGTGTTGCGGATGCCAGCGTTGTCCCGATTTCATTGTCGACGCATCTTATGGCGTCGACGTATGGAGTCGCAGAACAGGCTAGTAATATCATTCGTGCGCACTACACGGATAGTAGGACTACAGGCACGAGTAGTTCCGATCCTGGCTCTGCGTCGTCACCGACAAGCAGTGCATTGGGCGCTGAAGGGACTACTGGGGCGATTAGTGCTCATACAGCGCCTTCTGGTGGTGTACGAAGCGTTTCTGCGGTATCCGCTTGGGTTGCTGTTGTGTTCGCTGCAGCTGTTTCCATTTTCCATTCCTTGCATTGA (SEQ ID NO: 16)MRLGSIGAGLALLAALAVLAAHVHALAPRTQIAEEYDFVVVGGGQAGLVIGARLSEIANYTVLVLEAGTNGDEFRERIGTYNFYTPAYSYYESLWTTPMNWAYYTVPQSHAENRQIEWTRGKGLGGSSAINGLYLTRPGKEEINAWKDLLGDMDGADNWSWDSFYAAMKKSETFTPPSNEIATEGNITWDLSTRGIQGPIQATYPGYTFPQVGEWVMSLEAMGIASSNDMYGGEVYGAEVSTSSINPTNWTRSYSRTGYLDPLADNGNYDVVADAFVTRILFDASSPSNNLTANGVQYTLDNGKTNCTVKVKKEVILSAGTVGSPAVLLHSGVGPKDVLSDAGVELVSELPGVGHHLQDHFNNTLYLSYIDSAIAYINSTLMYGDNLDALQKNITTQINQFVLNTTYDAGVIAGYKAIANMTATTILSSSIGQIELLFMNSDLNGDIGITAALQHPYSHGRIYINSSNPLDYPVIDPNYLAVSADYEILRDGLNLARQLGNTQPLSSCLIAETIPGPSVKTDDDWLEWIREATGTEFHPSSSCAMLPREQGGVVDANLRVYGLANVRVADASVVPISLSTHLMASTYGVAEQASNIIRAHYTDSRTTGTSSSDPGSASSPTSSALGAEGTTGAISAHTAPSGGVRSVSAVSAWVAVVFAAAVSIFHSLH

Example 1 Fungal Strains and Methods

As described below, variants of fungal strain C1 were prepared. Inaddition, the Trichoderma reesei cellulase enzyme mixture (“Turbo”) usedin the following Examples is produced by a strain modified to produceand secrete high levels of the TrCel3A beta-glucosidase, encoded by T.reesei bgl1, as described in U.S. Pat. No. 6,015,703.

Strains and Nomenclature

Strain CF-400 (Δcdh1) is a derivative of C1 strain(“UV18#100fΔalp1Δpyr5”) further modified with a deletion of cdh1,wherein cdh1 comprises the polynucleotide sequence of SEQ ID NO:5.Strain CF-401 (Δcdh1Δcdh2), is a derivative of the C1 strain furthermodified with a deletion of both a cdh1 and a cdh2, wherein cdh2comprises the polynucleotide sequence of SEQ ID NO:7. Strain CF-402(+Bgl1) is a derivative of the C1 strain further modified foroverexpression of an endogenous beta-glucosidase 1 enzyme (Bgl1). StrainCF-403 is a derivative of the C1 strain modified with a deletion of cdh1and further modified to overexpress bgl1. Strain CF-404 is a derivativeof the C1 strain further modified to overexpress bgl1 with a deletion ofboth cdh1 and cdh2.

Cellulolytic enzymes from strain CF-400, CF-401, CF-402, CF-403 andCF-404, were produced by submerged liquid culture fermentation usingmethods well-known in the art.

The T. reesei Turbo cellulose was produced by submerged liquid culturefermentation using methods described in U.S. Pat. Appln. Publ. No.2010/0304438. The filtered fermentation broth was desalted using Biospincolumns (Biorad) following the manufacturer's protocol. Total proteinconcentration of the desalted enzyme was assayed using a BCA kit (Sigma)with a bovine serum albumin (Sigma) control.

Hydrolysis Reaction Conditions

Wheat straw (“WS”) was pretreated using the methods described in U.S.Pat. No. 4,461,648. Following pretreatment, sodium benzoate was added ata concentration of 0.5% as a preservative. The pretreated material wasthen washed with six volumes of lukewarm (˜35° C.) tap water using aBuchner funnel and filter paper to produce the substrate for subsequenthydrolysis reactions (“pretreated WS”).

The cellulosic portion of the pretreated WS was hydrolyzed using thecellulolytic enzyme systems obtained as described above. Pretreated WSwas hydrolyzed with 30 mg of cellulase per g of cellulose in reactionswith 50 g/L cellulose at 50° C. and pH 5.0, with 250 rpm orbitalshaking, in total reaction volumes of 50 mL unless specified. Forhydrolysis reactions containing the cellulase enzyme mixtures producedby C1 strains CF-400 and CF-401, beta-glucosidase purified from theTurbo or CF-402 cellulases was added at a dose of 125 IU per gmcellulose.

Detection of Glucose Yield in Hydrolysis Reaction

In this reaction, 1 mL aliquots of reaction mixture were sampledperiodically from reaction flasks. Each reaction was well mixed duringsampling to avoid removing a disproportionate amount of solid orsupernatant. The reaction was stopped by incubating the aliquot in a100° C. hot block for at least 5 minutes. The supernatants of eachreaction were analyzed for glucose concentration to determine the extentof conversion. The conversion calculation included correction terms forthe effect of glucose on the density of the solution and the volumeexclusion effect of non-hydrolyzable lignin present in the reaction.Glucose concentration was determined using a coupled enzymatic assaybased on glucose oxidase and horseradish peroxidases using methods knownin the art (See e.g., Trinder, Ann. Clin. Biochem., 6:24-27 [1969]).

Detection of Cellulose Conversion

Residual solids from each of the 50 mL reactions were recovered, washedand analyzed by infrared spectroscopy. Aliquots of the samples werecentrifuged at 2500 rpm for 5 min in an Eppendorf microfuge to sedimentthe solids, the supernatant was decanted, and the solids wereresuspended back to the original volume in water. This procedure wasrepeated 5 times. Washed solids were placed on the detection crystal ofa Golden Gate ATR cell installed on a Bruker Vertex 70 infraredspectrometer and absorbance was measured between 500-4000 cm⁻¹.

Example 2 Purification of C1 CDH1

First, 400 mL of C1 supernatant was concentrated to 140 mL using arotary evaporator. Then, 63 mL of the concentrate was buffer-exchangedinto 20 mM MOPS buffer pH 7.0 using 4 in-line Hi-Prep 26/10 desaltingcolumns (GE Healthcare, 17-5087-02). The resulting buffer-exchangedsupernatant (˜150 g/L total protein) was loaded onto a column containing500 mL DEAE Fast Flow resin (GE Healthcare, 17-0709-01) pre-equilibratedwith 20 mM pH7.0 MOPS buffer. The column was rinsed with 1 column volume(CV) of 20 mM MOPS (pH7.0) and then a 0-300 mM sodium chloride gradientwas run over 12 column volumes. Fractions were collected and analyzed byNuPage® Novex® Bis-tris SDS-PAGE gels (Invitrogen, NP0322BOX). TheSDS-PAGE bands corresponding to the apparent molecular weight of CDH1were analyzed by MS (performed by Alphalyse). The mass-mapping analysisconfirmed the presence of CDH1 in late-eluting fractions. Fractionscontaining CDH1, as demonstrated by SDS-PAGE gel, and confirmed by MSwere pooled and concentrated by ultrafiltration using Sartoriuscentrifugal 10 kDa filter (Sartorius-Stedim, VS2002). Then, 10 mL 500 mMpiperazine (pH 5.6) and 45 mL saturated ammonium sulfate were added to45 mL of the CDH1-containing pool and the resulting mixture was loadedonto a Phenyl FF (high sub) 16/10 column (GE Healthcare, 28-9365-45)pre-equilibrated with 1.6 M ammonium sulfate in 50 mM piperazine, pH5.6. A gradient of 1.6 M to 0 M ammonium sulfate in 50 mM piperazine, pH5.6, was run over 30 CV. Fractions were collected and SDS-PAGE gelanalysis was performed on the selected fractions as described above,revealing CDH1 eluted in the final rinse step with approximately 80-90%purity.

CDH1 activity was measured using a DCPIP (2,6-dichlorophenolindophenol)reduction assay similar to that described by Schou et. al. (Schou etal., Biochem J., 330:565-71 [1998]). Briefly, In a UV-transparentflat-bottom 96-well plate, 50 μL CDH1-containing fractions were added to150 μL of a solution of 1.0 g/L cellobiose and 100 μM DCPIP in 100 mMsodium acetate, pH 5.0. Samples were agitated briefly at roomtemperature and then absorbance at 530 nm (A₅₃₀) was measured for 10minutes. C1 CDH1-containing fractions displayed a rapid drop inabsorbance at 530 nm. DCPIP assays were performed using varying amountsof glucose or cellobiose with purified CDH1. Serial dilutions ofcellobiose (1.0 g/L to 7.8 mg/L) and glucose (10 g/L to 78 mg/L) wereprepared in a 96-well shallow-well plate. 20 μL glucose and cellobiosestandards were added to 160 μL/well 200 mM DCPIP (in 100 mM pH 5.0sodium acetate). Reactions were initiated by addition of 20 μL CDH1solution. Absorbance at 530 nm was monitored for 30 minutes. Comparisonsof the rates of decrease in absorbance at 530 nm indicate that C1 CDH1is approximately 10-fold more active on cellobiose than glucose.

Example 3 Making of CDH1 Split Marker Deletion Constructs

Genomic DNA was isolated from the C1 strain using standard procedures.Briefly, hyphal inoculum was seeded into a growth medium and allowed togrow for 72 hours at 35° C. The mycelial mat was collected bycentrifugation, washed, and 50 μL DNA extraction buffer (200 mM Tris, pH8.0; 250 mM NaCl; 125 mM EDTA; 0.5% SDS) was added. The mycelia wereground with conical grinder, re-extracted with 250 μL extraction buffer,and the suspension was centrifuged. The supernatant was transferred to anew tube containing 300 μL isopropanol. DNA was collected bycentrifugation, washed twice with 70% ethanol, and diluted in 100 μL ofwater.

Genomic DNA fragments flanking the cdh1 gene were cloned using primerscf09067 and cf09068 (cdh1 upstream homology) and primers cf09069 andcf09070 (cdh1 downstream homology). PCR reactions were performed byusing the GoTaq® Polymerase (Promega) following the manufacturer'sinstructions using 0.2 μM of each primer. Amplification conditions were95° C. 2 minutes, followed by 35 cycles of 95° C. for 30 seconds, 55° C.for 30 seconds (for upstream homology) or 53° C. for 30 seconds (fordownstream homology), and 72° C. for 1 minute, and followed by finalextension at 72° C. for 5 minutes. The pyr5 gene was PCR amplified as asplit marker from a vector using primers cf09024 and cf09025 (for the 5′portion of the gene) and cf09026 and cf09027 (for the 3′ portion of thegene). PCR reactions were performed using the GoTaq® Polymerase(Promega) following the manufacturer's instructions using 0.2 μM of eachprimer. Amplification conditions were 95° C. for 2 minutes, followed by35 cycles of 95° C. for 30 seconds, 53° C. for 30 seconds, and 72° C.for 1 minute, followed by a final extension at 72° C. for 5 minutes.Primers are shown in Table 3-1. In separate strand overlap extensionreactions (Horton et al., Meth. Enzymol., 217:270-279 [1993]), the PCRproducts resulting from primers cf09067 and cf09068 and primers cf09026and cf09027 were fused as were the PCR products resulting from primerscf09069 and cf09070 and primers cf09024 and cf09025. PCR reactions wereperformed by using Finnzymes' Phusion® DNA Polymerases following themanufacturer's instructions including 3% DMSO and using 0.2 μM of eachprimer. Amplification conditions were 98° C. for 1 minute, followed by35 cycles of 98° C. for 10 seconds, 62° C. for 20 seconds, 72° C. for 2minutes, and followed by final extension at 72° C. for 5 minutes. Thestrand overlap extension products were used for cdh1 deletion.

TABLE 3-1 Primer Sequences Primer Name Sequence (5′-3′) cf09067CACGCGGGGTTCTTTCTCCATCTC (SEQ ID NO: 17) cf09068TGAGGAAAACGCCGAGACTGAGCTCGACTCTGCCGGCCT ACCTACGA (SEQ ID NO: 18) cf09069ATCAGTTGGGTGCACGAGTGGGTTTTGATGGGGAGTTGA GTTTGTGAA (SEQ ID NO: 19)cf09070 GGATGGATGAGGTTGTTTTTGAGC (SEQ ID NO: 20) cf09024AACCCACTCGTGCACCCAACTGAT (SEQ ID NO: 21) cf09025GACCACGATGCCGGCTACGATACC (SEQ ID NO: 22) cf09026ACATGGCCCCACTCGCTTCTTACA (SEQ ID NO: 23)

Example 4 Transformation Method

C1 cells and derivative strains were inoculated into 100 mL growthmedium in a 500 mL Erlenmeyer flask using 10⁶ spores/mL. The culture wasincubated for 48 hours at 35° C., 250 rpm. To harvest the mycelia, theculture was filtered over a sterile Myracloth filter (Calbiochem) andwashed with 100 mL 1700 mM NaCl/CaCl₂ solution (0.6 M NaCl, 0.27 MCaCl₂*H₂O). The washed mycelia were transferred into a 50 mL tube andweighed. Caylase (20 mg/gram mycelia) was dissolved in 1700 mMNaCl/CaCl₂ and UV-sterilized for 90 sec. Then 3 mL of sterile Caylasesolution was added into the tube containing washed mycelia and mixed.Then 15 mL of 1700 mM NaCl/CaCl₂ solution was added into the tube andmixed. The mycelium/Caylase suspension was incubated at 30° C., 70 rpmfor 2 hours. Protoplasts were harvested by filtering through a sterileMyracloth filter into a sterile 50 mL tube. 25 mL cold STC (1.2 Msorbitol, 50 mM CaCl₂*H₂O, 35 mM NaCl, 10 mM Tris-HCl) was added to theflow through and spun down at 2720 rpm (1500×g) for 10 min at 4° C. Thepellet was resuspended in 50 mL STC and centrifuged again. After thewashing steps the pellet was resuspended in 1 mL STC.

Into the bottom of a 15 mL sterile tube, 2 μg DNA of each pyr5::Δcdh1strand overlap extension product was pipetted and 1 μLaurintricarboxylic acid and 100 μL protoplasts were added. The contentwas mixed and the protoplasts with the DNA were incubated at roomtemperature for 25 min. 1.7 mL PEG4000 solution (60% PEG4000(polyethylene glycol, average molecular weight 4000 daltons), 50 mMCaCl₂.H₂O, 35 mM NaCl, 10 mM Tris-HCl) was added and mixed thoroughly.The solution was kept at room temperature for 20 min. The tube wasfilled with STC, mixed and centrifuged at 2500 rpm (1250×g) for 10 minat 4° C. The STC was poured off and the pellet was resuspended in theremaining STC and plated on minimal selective media plates, lackinguracil, but containing 0.67 M sucrose as an osmotic stabilizer. Theplates were incubated for 5 days at 35° C. Colonies were restreaked andchecked for the deletion of cdh1, designated as strain “CF-400.”

Example 5 Confirmation of CDH1 Deletion

Genomic DNA was prepared as described in Example 3. Primer pairs cf09112and cf09113 (PCR reactions were performed by using the GoTaq® Polymerase(Promega) following the manufacturer's instructions using 0.2 μM of eachprimer. Amplification conditions were 95° C. for 2 minutes, followed by35 cycles of 95° C. for 30 seconds, 54° C. for 30 seconds, 72° C. for 30seconds, and followed by final extension at 72° C. for 5 minutes) aswell as cf09110 and cf09111 (reactions were performed by using theGoTaq® Polymerase (Promega) following the manufacturer's instructionsusing 0.2 μM of each primer. Amplification conditions were 95° C. for2′, followed by 35 cycles of 95° C. for 30″, 55.4° C. for 30″, 72° C.for 30″, and followed by final extension at 72° C. for 5′) were used inseparate PCR reactions to confirm absence of the cdh1 gene. Primerscf09181 and cf09091 were used in PCR to confirm proper junctionstructure and targeting of the pyr5 marker construct (See, Table 5-1).The PCR reaction was performed by using the GoTaq® Polymerase (Promega)following the manufacturer's instructions using 0.2 μM of each primer.Amplification conditions were 95° C. for 2 minutes, followed by 35cycles of 95° C. for 30 seconds, 54.4° C. for 30 seconds, 72° C. for 3minutes 30 seconds, and followed by final extension at 72° C. for 5minutes. PCR products were run on agarose gel to confirm a bandingpattern indicative of cdh1 deletion.

TABLE 5-1 Primer Sequences Primer Name Sequence (5′-3′) cf09110AAGCGTGCCGATTTTCCTGATTTC (SEQ ID NO: 24) cf09111GCATTTCTGGGGCGGTTAGCA (SEQ ID NO: 25) cf09112TCATCGACGCCTCCATCTTCC (SEQ ID NO: 26) cf09113TTTCGGTTGTCGTGTTTCCATTAT (SEQ ID NO: 27) cf09181GGAGATCCTGGAGGATTTCC (SEQ ID NO: 28) cf09091CAGGCGGTGTGCGTTATCAAAA (SEQ ID NO: 29)

A colorimetric dichlorophenolindophenol (DCPIP) assay was used to testfor deletion of cdh1 in CF-400. Deletion of cdh1 was determined bydecreased ability to reduce the DCPIP substrate compared to a cellulaseenzyme mixture (or culture filtrate) produced by the parent strain.Cells of the parental C1 strain and putative cdh1 delete strain weregrown and supernatant tested for DCPIP activity. Combined in microtiterplates were 160 μL of freshly made DCPIP reagent solution (0.2 mM DCPIPin 100 mM sodium acetate, pH 5.0), 20 μL cellobiose solution (1 g/Lcellobiose in deionized water), and 20 mLs of undiluted cellsupernatant. The absorbance of the solution was immediately measuredover time at 530 nm in kinetic mode for 30 minutes to track loss ofabsorbance as a result of DCPIP reduction. Supernatant from strainsdisplaying decreased ability to reduce the DCPIP substrate were run onSDS-PAGE to confirm the absence of CDH1.

Proteins from culture supernatants of submerged liquid culturefermentations of CF-400 and the untransformed parent were separated bySDS-PAGE using standard protocols. The proteins were visualized bystaining with Simply Blue Safe Stain (Invitrogen), as per manufacturer'sinstructions. The Cdh1 protein was observed as a ˜90 kD band in theuntransformed parent but was absent in CF-400.

Example 6 Deletion of CDH2

Genomic DNA was isolated as described in Example 3. Genomic DNAfragments flanking the cdh2 gene were cloned using primers cf10340 andcf10341 (cdh2 upstream homology) and primers cf10342 and cf10343 (cdh2downstream homology). PCR reactions were performed by using the GoTaq®Polymerase (Promega) following the manufacturer's instructions using 0.2μM of each primer. Amplification conditions were 95° C. for 2 minutes,followed by 35 cycles of 95° C. for 30 seconds, 58° C. for 30 seconds(for upstream homology) or 58.4° C. for 30 seconds (for downstreamhomology), 72° C. for 1 minute, and followed by final extension at 72°C. for 5 minutes. The hygromycin gene was PCR amplified as a splitmarker from a vector using primers cf10176 and cf10177 (for the 5′region of the gene) and cf10178 and cf10179 (for the 3′ region of thegene). PCR reactions were performed by using the GoTaq® Polymerase(Promega) following the manufacturer's instructions using 0.2 μM of eachprimer. Amplification conditions were 95° C. for 2 minutes, followed by35 cycles of 95° C. for 30 seconds, 56.3° C. for 30 seconds, 72° C. for1 minute 30 seconds, and followed by final extension at 72° C. for 5minutes. Primers are shown in Table 5. In separate strand overlapextension reactions (Example 3) the PCR products resulting from primerscf10340 and cf10341 and primers cf10178 and cf10179 were fused as werethe PCR products resulting from primers cf10342 and cf10343 and primerscf10176 and cf10177. PCR reactions were performed by using Phusion® DNAPolymerases (Finnzymes) following the manufacturer's instructions andincluding 3% DMSO and 0.2 μM of each primer. Amplification conditionswere 98° C. 1 minute, 35 cycles of 98° C. 10 seconds, 62° C. 20 seconds,72° C. 2 minutes and final extension at 72° C. 5 minutes. The strandoverlap extension products were used for cdh2 deletion.

TABLE 5-1 Primer Sequences Primer Name Sequence (5′-3′) cf10340TTCAGCACGGCCGGGGATTTTATCCA (SEQ ID NO: 30) cf10341GTAACACCCAATACGCCGGCCGAACATAAGAGCGGAGGTCAG GAATAA (SEQ ID NO: 31)cf10342 CCGTCTCTCCGCATGCCAGAAAGAGCTGTCAACGCTGGTTTGTGGTGG (SEQ ID NO: 32) cf10343 AATGCCGGACCGCGAGTTCAGGTA (SEQ ID NO: 33)cf10176 TCTTTCTGGCATGCGGAGAGACGG (SEQ ID NO: 34) cf10177TGTTGGCGACCTCGTATTGGGAAT (SEQ ID NO: 35) cf10178TCTCGGAGGGCGAAGAATCTCGTG (SEQ ID NO: 36) cf10179TTCGGCCGGCGTATTGGGTGTTAC (SEQ ID NO: 37)

Strain CF-401 cells were grown and transformed as described in Example4. Transformed colonies of CF-401 were restreaked and checked fordeletion of cdh2.

Genomic DNA was prepared as described in Example 3. Deletion of cdh2 inCF-401 was confirmed by PCR. Primers cf10326 and cf10327 were used inPCR to confirm absence of the cdh2 gene. The PCR reaction was performedby using the GoTaq® Polymerase (Promega) following the manufacturer'sinstructions using 0.2 μM of each primer. Amplification conditions were95° C. for 2 minutes, followed by 35 cycles of 95° C. for 30 seconds,59.3° C. for 30 seconds, 72° C. for 30 seconds and followed by finalextension at 72° C. for 5 minutes. Primers cf10364 and cf10295 were usedin PCR to confirm proper junction structure and targeting of thehygromycin marker construct (Table 6-2). The PCR reaction was performedby using the GoTaq® Polymerase (Promega) following the manufacturer'sinstructions using 0.2 μM of each primer. Amplification conditions were95° C. 2 minutes, 35 cycles of 95° C. 30 seconds, 56° C. 30 seconds, 72°C. 3 minutes and final extension at 72° C. 5 minutes. PCR products wererun on agarose gel to confirm a banding pattern indicative of cdh2deletion. Dichlorophenolindophenol (DCPIP) assay and SDS-PAGEconfirmation of cdh2 deletion were performed as described in Example 5.Deletion of cdh2 from strain CF-401 was determined by decreased abilityof the resulting culture filtrate (or cellulase enzyme mixture) toreduce the DCPIP substrate compared to that produced by the parentstrain CF-400.

TABLE 6-2 Primer Sequences Primer Name Sequence (5′-3′) cf10326GCGCTGGAAAAGGATGCCACCGAGT (SEQ ID NO: 38) cf10327GCACCCCACTGTCCGAAACCGTTA (SEQ ID NO: 39) cf10295AGCGCGTCTGCTGCTCCATACAAG (SEQ ID NO: 40) cf10364CAAAGCCACGTCCAGGTTGATAGA (SEQ ID NO: 41)

Example 7 Hydrolysis of Pretreated Wheat Straw by an Enzyme MixtureLacking CDH1

The ability of CF-402 and CF-403 enzyme mixtures to saccharify thecellulose present in pretreated wheat straw (WS) as measured by glucoseand gluconate production was compared in a hydrolysis assay as describedin Example 1. Each enzyme mixture was assessed using both a singleenzyme dose of 30 mg enzyme/g cellulose and in parallel using a second30 mg enzyme/g cellulose dose added after a 24 hr hydrolysis to theoriginal 30 mg/g enzyme load. The preparation of pretreated WS used inthese experiments had a maximum convertibility of 95%; conversions ofall enzymes were normalized. Hydrolysis assay results are depicted inFIG. 1.

In the single dose experiment, the total soluble products measured,equaled a cellulose conversion of 79% and 87% of theoretical maximumglucose yield with enzyme mixtures from CF-402 and CF-403 respectively.About 5% of soluble products were measured to be gluconate for CF-403 ascompared to 10% for CF-402. The results demonstrate that removing theCDH1 enzyme from the cellulase mixture produced by C1 derived strainsimproves glucose yield.

In the multidose experiments, the total soluble products measured withenzyme mixtures from CF-402 indicated a 90% cellulose conversion ascompared to 95% with enzyme mixtures from CF-403. The conversion ofglucose to gluconate was higher for CF-402 (14%) compared to CF-403(7%).

Example 8 Hydrolysis of Pretreated Wheat Straw by an Enzyme MixtureLacking Both CDH1 and CDH2

The ability of enzyme mixtures to saccharify the cellulose present inpretreated WS was compared for enzyme mixtures obtained from CF-401,CF-400, and CF-402 in the following hydrolysis assay.

Enzyme mixtures derived from CF-400, CF-401 and CF-402 were produced byfermentation as described in Example 1. Enzyme mixtures derived fromCF-400, CF-401 and CF-402 were added to the pretreated wheat straw atenzyme loads of 3.83-6% w.r.t cellulose. The cellulose concentration was110 g/L. Aspergillus niger beta-glucosidase (ANBG, Sigma) at an enzymeload of 2% w.r.t cellulose, was supplemented to CF-400 and CF-401 enzymemixtures as these strains lack added beta-glucosidase activity. Glucoseand gluconate yields were compared for the sample withdrawn at 48-70hrs.

For glucose analysis, the samples were diluted 1:10 using 10 mM H₂SO₄and then analyzed using an Agilent HPLC 1200 equipped with HPX-87H Ionexclusion column (300 mm×7.8 mm) with 5 mM H₂SO₄ as a mobile phase at aflow rate of 0.6 mL/min at 65° C. The retention time of the glucose was9.1 minutes. The gluconate analysis was carried out using LC-MS. TheLC/MS/MS used was a API2000 triple quadrupole system (AB Sciex) equippedwith Agilent 1100 HPLC, and CTC PAL autosampler. The column used was aHYPERCARB 50×2.1 mm, 3 μm at 80° C. temperature. The chromatographymethod was a 2 minutes isocratic (95% A) run with a flow rate of 350μL/min. The two mobile phases contained 1.5% NH₄OH. The mobile phase Awas aqueous, and the B phase was a 50:50 solution of CH₃CN:IPA. TheMS/MS transition monitored for gluconate was 194.99/161.10. Theanalytical methods and controls for measuring gluconate and glucose wereslightly different for CF-402. However, the methods and controls usedare well known in the art.

The results are provided in FIG. 2 and in Table 7-1 below.

TABLE 7.1 Glucose Conversion Fractional Conversion of AvailableCellulose Enzyme Mixture Glucose Gluconate Sum Bgl1 0.800 0.167 0.967(CF-402) Δcdh1 0.861 0.114 0.974 (CF-400) Δcdh1Δcdh2 0.954 0.024 0.978(CF-401)

Results from the experiments show the total soluble products obtainedfrom the enzyme mixtures derived from strains CF-400, CF-401 and CF-402were similar. However the ratios of glucose:gluconate were different inthe soluble products produced by the enzyme mixtures from the differentstrains. CF-402-derived enzyme mixtures produced about 23 g/L ofgluconate. In comparison, CF-400-derived enzyme mixtures (the deletionof the cdh1 gene) reduced the gluconate production to about 16 g/L. Theenzyme mixture resulting from the additional deletion of the cdh2 genein CF-401 significantly reduced (3.3 g/L) the gluconate production. Thisreduction in gluconate production represented an 86% reduction comparedto the enzyme mixtures derived from CF-402 strain. Correspondingly theglucose yields increased from 112 g/L with CF-402 to 133.6 g/L withCF-401-derived enzyme mixtures. The results demonstrate that the removalof CDH1 and CDH2 enzymes by deletion of both cdh1 and cdh2 genessignificantly increases glucose yield with a concomitant decrease ingluconate production in the saccharification reaction.

Example 9 Enzymatic Hydrolysis of Cellulose

The cellulosic portion of pretreated WS was hydrolyzed according to themethods described in Example 1 using enzyme mixture derived from CF-402.In each reaction, an initial 30 mg/g dose of CF-402 was allowed to reactwith the pretreated WS substrate. After 24 hours, an additional dose of30 mg/g of CF-402 enzyme mixture was added to one reaction flask.

Aliquots of 1 mL were sampled periodically from all these flasks, andglucose concentrations were determined using a coupled enzymatic assayas described in Example 1. By glucose yield, a single 30 mg/g dose ofthe CF-402 enzyme mixture produces 69.5% of theoretical maximum glucoseyield (Gmax). Dosing additional CF-402 increased this yield to 75.6% asshown in FIG. 11.

Example 10 Conversion of Glucose by C1-Derived Enzyme Mixtures

Fifty-two mg of the C1-derived CF-402 enzyme mixture or the T. reesei“Turbo” enzyme mixture was mixed with a solution containing both 50% w/wglucose and 5% w/w cellobiose or a solution containing 50% w/w glucosealone, at pH 5.0 and 60° C. for 24 hr and 48 hr.

An unknown species (detected in HPLC chromatograms as described in moredetail herein) was produced in a time-dependent manner in thesereactions (Table 2). The peak areas of the unknown species arenormalized to the peak area of the unknown species in the 24 hr reactionof the CF-402 enzyme mixture and glucose only. These data indicate thatthe insoluble cellulose substrate, primarily comprising cellulose andlignin, need not be present for the formation of this species. Thepresence of glucose alone was sufficient for the formation of thespecies, although production of the species was enhanced by the additionof cellobiose.

TABLE 10-1 Production of Unknown Compound by Enzyme Mixtures Peak Areaof Unknown [Glucose] [Cellobiose] (normalized) (w/w) (w/w) Enzyme 24 h48 h 50% — Turbo 0.08 0.09 CF-402 1.00 1.44 5% Turbo 0.13 0.14 CF-4021.32 1.70

Example 11 Analysis of Cellulose Hydrolysis Products Using ConcentratedAcid Hydrolysis

The unknown species observed in Example 10 was subjected to acidconditions known to hydrolyse oligosaccharides to test if it representeda glucose oligomer. Such a glucose oligomer could be present as either adirect product of cellulose hydrolysis or from synthesis reaction of thedirect products through a reaction such as transglycosylation.Cellotriose, a beta 1-4 trimer of glucose, was used as a positivecontrol. Acid hydrolysis was performed by mixing an equal volume of asample and 98% sulphuric acid. Acid hydrolysis completely abolished thecellotriose peak in a HPLC chromatogram collected using the methoddescribed herein (FIG. 12A) but it did not significantly alter the peakarea of the unknown species, though it did have a minor effect on itsretention time (FIG. 12B). These results indicate that the unknownspecies is not a glucose oligomer.

Example 12 Infrared (IR) Spectroscopy of Lignocellulosic Hydrolysate

IR spectroscopy was used to analyze the hydrolysate from reactions withpretreated wheat straw in Example 11. Hydrolysates were selected fromreactions which had reached their endpoint (i.e. no additional glucoseformation was observed). Hydrolysate was applied dropwise to the ATRdetection crystal of a Bruker Vertex 70 Infrared spectrometer, and eachdrop was allowed to evaporate to form a cast film. A peak at awavenumber of 1715 cm⁻¹ in the spectrum of CF-402 hydrolysate was notobserved in the Turbo hydrolysate (FIG. 13). This wavenumber isassociated with a vibrational mode of a carbonyl group. In conjunctionwith other observations described herein that the unknown species can beproduced by the CF-402 enzyme mixture from glucose alone, this indicatesthat the unknown species is an oxidized form of glucose.

Example 13 Identification and Quantification of Glucono-Lactone inEnzymatic Cellulose Hydrolysates

There are three forms of oxidized glucose: gluconic acid formed byoxidation at carbon 1, glucuronic acid formed by oxidation at carbon 6and glucaric acid which is oxidized at carbons 1 and 6. Gluconic acidexists in pH-dependent equilibrium with its lactone form (gluconolactoneor 1,5-gluconolactone, or D-glucono-1,5-lactone) produced viaesterification. High-performance liquid chromatography (HPLC) wasperformed to characterize these compounds in enzymatic cellulosehydrolysates.

Glucuronic acid, glucaric acid and the lactone form of gluconic acidwere analyzed by anion-exchange HPLC. The HPLC system was a DionexDX-500 modular chromatography system consisting of a GP50 gradient pump,a Rheodyne (Idex) six port sampling valve, an AS40 automated sampler, anED40 or ED50 electrochemical detector with a gold working electrode andAg/AgCl reference electrode for pulsed amperometric detection (PAD). TheCarboPac™ PA1 column (4×250 mm; Dionex) and guard (4×50 mm) consist of a10 μm diameter polystyrene/divinylbenzene substrate agglomerated with580 nm MicroBead quaternary ammonium functionalized latex (2% crosslinkage; Microbeads).

Samples of glucuronic acid, glucaric acid and gluconolactone werediluted in water to bring them within range of the standards(0.003-0.030 g/L) and injected in a volume of 25 μL. Samples weresubjected to an isocratic separation in a solution of 6 mM NaOH for 28minutes after injection. A 6 minute gradient, raising the NaOHconcentration linearly from 10 mM to 300 mM, was then applied. Next, a 5minute gradient, raising the NaOH concentration from 300 mM to 1 M, wasapplied. The column was then re-equilibrated with 6 mM NaOH prior to thenext run. Peak integration and quantification of standards and unknownswas performed with CHROMELEON® software (Dionex).

Glucose was mixed with a secreted enzyme mixture harvested from CF-402.Specifically, fifty-two mg of the CF-402 enzyme mixture was mixed with asolution containing both 50% w/w glucose, at pH 5.0 and 60° C. for 24 hrand 48 hr. The product of this reaction was analyzed using the aboveHPLC conditions. One component from the product of this reactionco-eluted with both glucaric acid and gluconolactone (FIG. 14A).

A second HPLC method was used to analyze the glucaric acid, gluconicacid lactone, and the product of the reaction of glucose with thesecreted enzyme mixture harvested from CF-402. This method employs aIonPac®AS11-HC column (4×250 mm; Dionex) and AG11-HC guard (4×50 mm)consisting of a 9 μm diameter ethylvinylbenzene polymer cross linkedwith 55% divinylbenzene polymer agglomerated with a 70 nm alkanolquaternary ammonium latex (6% latex cross linkage) and a capacity of 290μeq/column (4×250 mm). The HPLC system used for this method comprised adual gradient pump (DP) with vacuum degassing, dual Rheodyne (Idex) sixport sampling valves and an AS automated sampler with diverter valve.The system was equipped with both electrochemical and conductivitydetectors. An anion trap column (ATC) was installed in-line between theserial pump and the DC injection valve to remove any anionic species inthe eluent and a carbonate removal device which was installed betweenthe chemical suppressor and the conductivity detector to partiallyremove any carbonate in the mobile phase.

Samples of glucaric acid, gluconic acid lactone, and the product of thereaction of glucose with the secreted enzyme mixture harvested fromCF-402 were diluted in water to bring them within range of the standards(0.003-0.030 g/L) and injected in a volume of 25 μL. The samples weresubjected to an isocratic separation in a solution of 1 mM NaOH for 6minutes after injection. A six-minute gradient, raising the NaOHconcentration linearly from 1 mM to 60 mM was then applied. The columnwas subsequently cleaned with a short 1 M NaOH step prior torequilibration in 1 mM NaOH. Peak integration and quantification ofstandards and unknowns was performed with CHROMELEON® software (Dionex).

Using this second HPLC method, gluconolactone, but not glucaric acid,co-elutes with a component of the product of the reaction of glucosewith the secreted enzyme mixture harvested from CF-402 (FIG. 14B).Therefore, the unknown species is gluconolactone, which is produced bythe action of one or more enzymes within the C-1 enzyme system.

Example 14 Decrease in Glucose Yield from Cellulose Hydrolysis in thePresence of Oxidoreductases

Wheat straw was pretreated and washed as described in Example 1. Thecellulose in this pretreated material was hydrolysed by 30 mg T. reeseiTurbo cellulase per gram of cellulose. The reaction was performed at 50°C. and pH 5.0, with 250 rpm orbital shaking.

Enzymes from the E.C. 1 group of enzymes, the oxidoreductases, wereadded to the hydrolysate of this reaction in separate reactions. Glucoseoxidase from Aspergillus niger (E.C. 1.1.3.4), pyranose oxidase fromCoriolus sp. (E.C. 10.1.3.10) and glucose dehydrogenase from Pseudomonassp. (10.1.1.47), all purchased from Sigma-Aldrich, were tested.Enzymatic reactions were carried out over 72 h at the pH and temperatureoptima of each enzyme: pH 8.0 and 37° C. for glucose oxidase and glucosedehydrogenase; pH 7.0 and 37° C. for pyranose oxidase. For glucosedehydrogenase, a supplement of one equivalent (with respect to glucose)of beta-nicotinamide adenine dinucleotide hydrate (Sigma) was added tothe reaction mixture. Enzymes were dosed at 0.45 mg per gram of startingcellulose. This dose corresponds to 1.5% of the full dose of 30 mg/g,simulating the presence of a low-abundance oxidative enzyme in a mixedenzyme system. The glucose concentrations were determined in eachreaction flask and in no-enzyme controls by HPLC (Dionex ICS5000 with aCarboPac™ PA1, 4×250 mm column (Dionex), with an eluent of 6% 200 mMNaOH in degassed ddH₂O, run time of 25 minutes at a rate of 1.5 ml/minin which glucose retention time is 11.2 minutes) to determine theglucose yield loss. P-values were determined from a t-statisticcalculated for the case of unequal sample sizes and equal variances;there were two to four plus-enzyme and one to two no-enzyme flasks.

TABLE 14-1 Glucose Yield Loss Due to Enzymatic Action of OxidoreductasesEnzyme Glucose Yield Loss P-value Glucose Oxidase 4.38% <0.01 PyranoseOxidase 3.81% <0.01 Glucose Dehydrogenase 2.27% <0.05

These data indicate that a significant decrease in glucose yield can beproduced by oxidoreductases present in very low abundance in acellulolytic enzyme system. Elimination or reduction of these enzymaticactivities by any means would therefore result in improved glucoserecovery.

Example 15 Saccharification of Acid-Pretreated Corn Stover

In this Example, experiments conducted using CF-404 on acid-pretreatedcorn stover are described. In these experiments, 0.81% to 6% CF-404(with regard to glucan concentration) was added to a mixture of waterand pre-treated corn stover (NREL) (90 g/kg glucan) at a celluloseconcentration of 9% and pH 5. The mixture was incubated at 55° C., withshaking for 73 hours. During this incubation, samples were periodicallytaken and the sugar concentration determined using by HPLC, usingmethods known in the art. Cellulose and residual xylan were found tohave been converted to their monomeric sugars in high yield.

1. A Myceliophthora thermophile fungal cell that has been geneticallymodified to reduce the amount of endogenous cellobiose oxidizing enzymeactivity that is produced by the fungal cell, wherein the fungal cellhas been genetically modified by deleting endogenous cellobiosedehydrogenase enzymes having at least about 90% or at least about 95%sequence identity to the sequences of SEQ ID NOS:6 and
 8. 2. The fungalcell of claim 1, wherein the fungal cell has been genetically modifiedto reduce the amount of endogenous cellobiose dehydrogenase produced bythe fungal cell and to increase the production of at least onesaccharide hydrolyzing enzyme, wherein said at least one saccharidehydrolyzing enzyme is selected from beta-glucosidases, endoglucanases,cellobiohydrolases, esterases, beta-xylosidases, xylanases,arabinofuranosidases, alpha-glucuronidases, acetylxylan esterases,feruloyl esterases, alpha-glucuronyl esterases, and GH61 enzymes.
 3. Thefungal cell of claim 2, wherein said at least one saccharide hydrolyzingenzyme is a Myceliophthora thermophila enzyme.
 4. The fungal cell ofclaim 2, wherein said at least one saccharide hydrolyzing enzyme isoverexpressed by said fungal cell.
 5. The fungal cell of claim 2,wherein said overexpressed saccharide hydrolyzing enzyme isbeta-glucosidase.